The authors have no acknowledgements.

NOTE: All procedures involving the use of animals are carried out according to the local legislation. Our work is approved by the Sydney Local Health District Animal Welfare Committee. The allowed procedures are described in the project license documentation and follow guidelines that ensure the welfare of the animal at all times.&#160;Ensure adherence to the legislation on animal experimentation of the country where the work is performed.
1. Protocol for Preparing EM Fixative
<ol>
	<li>For 100 ml of fixative, prepare&#160;paraformaldehyde by adding 2 g of paraformaldehyde powder to 25 ml distilled water in a conical flask, heat to 65 &#186;C, stirring constantly with a magnetic stirrer. 
	NOTE: Glutaraldehyde, paraformaldehyde and sodium cacodylate buffer are all hazardous substances and must always be handled in fumehood.</li>
	<li>Heat at 65 &#186;C for 2 min then allow to cool. If needed, add sodium hydroxide solution dropwise while stirring to clear solution completely. 
	NOTE: sodium hydroxide is hazardous, handle with care.</li>
	<li>Add 10 ml of EM grade stock&#160;glutaraldehyde, 50 ml of 0.2 M sodium cacodylate buffer and 2 g of sucrose and stir with magnetic stirrer. Add 2 ml of 1.0 M CaCl2.</li>
	<li>Adjust to pH 7.4 prior to making solution up to 100 ml with distilled water. The final fixative contains 2.5 % glutaraldehyde, 2 % Paraformaldehyde, 2 mmol CaCl2, 2 % Sucrose in 0.1 M&#160; cacodylate buffer. Ensure that the total Osmolality is 440 mOsmol/L. Use within 24 hr&#160;of preparation.</li>
</ol>
2. Perfusion Fixation of Liver
<ol>
	<li>Warm perfusion buffer and fixative to 35 - 37 &#186;C. The correct temperature of the exsanguination buffer and fixative helps to ensure tissue integrity.</li>
	<li>Anaesthetize the animal with a single intraperitoneal injection of 50 mg/kg Ketamine and 5 mg/kg Xylazine.</li>
	<li>Once the animal is under deep anaesthesia, assessed by hind limb withdrawal and tail pinch, a Y incision is made with blunt ended scissors in the animal&#8217;s abdomen to expose the liver and portal vein. 
	NOTE: Ensure that equipment is clean to avoid contamination of specimens with microorganisms and general debris which will be readily seen on SEM.</li>
	<li>Tie two very loose sutures around the portal vein one proximal to the liver, the other more distally to the liver.</li>
	<li>Cannulate the portal vein with an appropriately sized IV cannula (18 G for rats, 22 G for mice).Tighten sutures to secure cannula.</li>
	<li>Commence perfusion with phosphate buffered saline warmed to 37 &#186;C, and at a pressure of 10 cm of H20. Between 5 and 20 ml of PBS is usually required to exsanguinate the liver. 
	NOTE: Do not to allow air bubbles to enter the liver through the tubes connected to the cannula as they induce artefacts secondary to embolization and pressure; Damage to the hepatocytes, sinusoids and LSEC fenestrations occurs if the perfusion pressure is too high.</li>
	<li>Sever the abdominal and thoracic vena cava to allow the buffer to exit freely from the liver. This prevents high back-pressure damage to LSECs.</li>
	<li>Once the liver is free of blood, replace PBS with EM fixative and perfuse for approximately 5 minutes, until the liver is hardened and very pale.</li>
	<li>Discontinue perfusion and cut the fixed liver into 1 - 2 mm3 blocks using a sharp scalpel.</li>
	<li>Post-fix tissue in EM fixative for 24 - 72 hr at 4 &#186;C. 
	NOTE: In general, underfixation causes more artefacts than overfixation.</li>
	<li>Change fixative following post-fix period to 0.1 M sodium cacodylate buffer for storage at 4 &#186;C. 
	NOTE: Specimens stored in this way will be preserved for a significant period of time (up to 12 months), however timely secondary fixation and examination is recommended for best results.</li>
</ol>
3. Needle Perfusion
NOTE: Needle fixation is a variant of perfusion fixation that involves injection of fixative directly into biopsied liver tissue. The aim is for fixative to be flushed through the blood vessels in order to exsanguinate them and expose all of the tissue block to fixative. Care must be taken to keep the injecting pressure low to avoid pressure injury18,19.
<ol>
	<li>Remove a small piece of liver from the animal or subject and rinse in saline to remove blood and debris.</li>
	<li>Draw the normal saline into syringe fitted with a fine gauge needle (typically an insulin syringe).</li>
	<li>Inject the saline very slowly into the liver until blood is flushed out of the tissue. Multiple injections may be required.</li>
	<li>Draw the EM fixative into another syringe fitted with a fine gauge needle (typically an insulin syringe).</li>
	<li>Inject the fixative very slowly into the liver until it becomes hard and tan in colour. Multiple injections may be required.</li>
	<li>Cut the fixed liver into 1 - 2 mm3 blocks using a sharp scalpel. Incubate in EM fixative for 24 - 72 hr 4 &#176;C. Wash 3 times in 0.1 M sodium cacodylate buffer before commencing secondary fixation.</li>
</ol>
4. Preparation for Scanning Electron Microscopy
<ol>
	<li>Wash sample 3 times in 0.1 M sodium cacodylate buffer. To fix lipids, post fix the sample in 2% osmium tetroxide in 0.1 M sodium cacodylate buffer for 2 hr. 
	NOTE: Complete washing is very important as glutaraldehyde and osmium tetroxide cross react and generate artefacts on SEM.</li>
	<li>Rinse the samples in increasing concentrations of ethanol to commence dehydration. First rinse in 50 % ethanol for 5 minutes; then 3 times for 5 min with 70% ethanol; rinse 3 times for 5 min with 90% ethanol; rinse 2 times for 5 min in 100 % ethanol; and finally rinse 2 times for 10 min in 100% ethanol (molecular sieve).</li>
	<li>Remove all ethanol from the samples and replace with hexamethyldisilazane (HMDS) in fumehood and leave for 10 min. Remove HMDS from the tissue samples and allow to evaporate. 
	NOTE: HMDS is highly hygroscopic when cold therefore allow to reach RT before using for final dehydration step.</li>
	<li>Optionally, mount the samples immediately or place them into a desiccator until ready to mount for SEM.</li>
</ol>
5. Mounting
NOTE: Correct specimen mounting will maximize the number of clearly delineated sinusoids available for analysis under SEM.
<ol>
	<li>Label the base of the metal SEM stubs and apply double-sided conductive carbon tape to the top of the stubs.</li>
	<li>Visualize samples using a dissecting microscope to identify the surface with the best sinusoids for subsequent SEM. Place that surface upwards onto the carbon tape surface of the stub.</li>
	<li>Stick specimens firmly to the stub, specimens are now ready for sputter coating.</li>
</ol>
6. Coating
NOTE: Coating the specimen with a fine film of conductive metal (gold or platinum) in a sputter coater grounds the specimen and protects it from damage from the electron beam. If the coating is too thick structures of interest may be obscured.
<ol>
	<li>Place stubs into the vacuum chamber of automatic fine sputter coater, set mode to rotation and automatic sputter coating for 45 sec. Use platinum coating for finer detailing, however gold is also suitable.</li>
	<li>Apply a thin layer of carbon paint to the external surfaces of the mounted liver specimens, taking care not to coat the sinusoidal surfaces.</li>
	<li>Once the specimens are coated with platinum and carbon paint has dried they are ready for observation on the SEM. Samples can be stored in a desiccator.</li>
</ol>
7. Using the Scanning Electron Microscope
<ol>
	<li>Place specimens into scanning electron microscope and return microscope to vacuum.</li>
	<li>Using standard microscope operating procedures for visualisation of specimens by commencing at a low magnification, gradually increasing magnification.</li>
	<li>Ensure the microscope is adjusted appropriately for working distance, spot size and astigmatism.</li>
	<li>Check for fixation integrity. If the LSEC is mostly free from gaps and contains fenestrations measuring 30 - 250 nm diameter this would generally indicate that specimen preparation has been successful.</li>
	<li>If the sinusoids are full of gaps or devoid of fenestrations the specimen preparation has not been successful or there is significant pathology. In this case, slice the sample with a very fine razor blade, and the cut surfaces recoated and analysed for successfully fixed sinusoids.</li>
	<li>At approximately 15,000-20,000 &#215; magnification select flat LSEC surfaces that are free of debris with good fixation and where the fenestrations are clearly visible (typically containing at least 10 fenestrations).</li>
	<li>Save images of at least 10 sinusoids per liver, from different areas of the blocks, using at least two blocks per liver. Sampling 10 micrographs at 15,000 - 20,000 &#215; magnification per animal is generally sufficient for the quantification of fenestrations to be representative of the entire liver.</li>
</ol>
8. Analysis
NOTE: ImageJ software which can be downloaded free from the NIH is utilized to quantify fenestration diameter and frequency (<a href="http://imagej.nih.gov/ij">www.imagej.nih.gov/ij</a>).
<ol>
	<li>Open Image J and set the scale using the scale bar embedded in the picture (Screen shot 1).</li>
	<li>Using the polygon tool in ImageJ, trace around the entire flat area of the LSEC including fenestrated and defenestrated areas. Don&#8217;t just trace the sieve plates as this will falsely inflate porosity. Exclude any large pieces of debris that are on the cell surface that might be obscuring fenestrations.</li>
	<li>To measure fenestration diameter, trace a line through the longest diameter of each fenestration by selecting the line tool, and press &#8220;m&#8221; to measure the line, and &#8220;d&#8221; (draw) to permanently draw the line on the picture. This line is defined as the fenestration diameter. The length of the line will now be available in the results box of ImageJ (Screen shot 2).</li>
	<li>Measure all gaps which are larger holes in the LSEC cytoplasm over 250 nm, as well as fenestrations. Gaps are often artefacts reflecting poor perfusion or fixation, however gaps also can occur as a result of disease and toxicity. The data for gaps will be excluded from calculation of fenestration parameters later in the analysis.</li>
	<li>The area of gaps that are not circular should be calculated by tracing around the gaps and calculating their area on ImageJ.</li>
	<li>Paste the data from the results box in ImageJ into an EXCEL spreadsheet for further analysis.</li>
</ol>
9. Calculations
<ol>
	<li>Average fenestration diameter = average of all fenestration diameters (not including gaps, where gaps are &#8805; 250 nm).</li>
	<li>Fenestration area = &#960;r2,&#160;where r, the radius, is calculated from the individual fenestrations diameter (r=d/2), not including gaps.</li>
	<li>Porosity (%) = (&#931; (&#960;r2)/total area analyzed -&#160;&#931; (area of gaps&#160; =)) (&#181;m2)) &#215; 100.</li>
	<li>Fenestration frequency = total number of fenestrations/ (total area analyzed-&#931; (area of gaps) (&#181;m2).</li>
</ol>
10. Presentation of Fenestration Data
NOTE: Whenever possible, publications including quantification of fenestration data should include the following information
<ol>
	<li>Fenestration diameter, with a statement confirming what boundary diameters where used to define fenestrations (typically between 50 - 250 nm), Fenestration frequency (number/&#181;m2) and porosity (%).</li>
	<li>A statement confirming whether gaps have been included in the analysis, frequency distribution graph of fenestration diameter and the number of livers, blocks, images and fenestrations should be included in the analysis.</li>
</ol>

<ol>
	<li>Cogger, V. C., Le Couteur, D. G., Arias, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Liver: Biology and Pathobiology. , 387-404 (2009).</li><li>Fraser, R., Dobbs, B. R., Rogers, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+and+the+liver+sieve:+the+role+of+fenestrated+sinusoidal+endothelium+in+lipoprotein+metabolism,+atherosclerosis,+and+cirrhosis.">Lipoproteins and the liver sieve: the role of fenestrated sinusoidal endothelium in lipoprotein metabolism, atherosclerosis, and cirrhosis.</a> Hepatology. 21, 863-874 (1995).</li><li>Wisse, W., De Zanger, R. B., Charels, K., Van Der Smissen, P., McCuskey, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+liver+sieve:+considerations+concerning+the+structure+and+function+of+endothelial+fenestrae,+the+sinusoidal+wall+and+the+space+of+Disse.">The liver sieve: considerations concerning the structure and function of endothelial fenestrae, the sinusoidal wall and the space of Disse.</a> Hepatology. 5 (4), 683-692 (1985).</li><li>Reilly, J. N., Cogger, V. C., Fraser, R., Le Couteur, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+feeding+and+fasting+on+fenestrations+in+the+liver+sinusoidal+endothelial+cell.">The effect of feeding and fasting on fenestrations in the liver sinusoidal endothelial cell.</a> Pathology. 42 (3), 255-258 (2010).</li><li>Tian, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+serotonin+receptor-2B+rescues+small-for-size+liver+graft+failure+in+mice.">Activation of serotonin receptor-2B rescues small-for-size liver graft failure in mice.</a> Hepatology. 53 (1), 253-262 (2011).</li><li>Cogger, V. C., Mitchell, S. J., Warren, A., de Cabo, R., Le Couteur, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-Related+Loss+of+Responsiveness+to+2,5-Dimethoxy-4-Iodoamphetamine+in+Liver+Sinusoidal+Endothelial+Cells.">Age-Related Loss of Responsiveness to 2,5-Dimethoxy-4-Iodoamphetamine in Liver Sinusoidal Endothelial Cells.</a> J Gerontol A Biol Sci Med Sci. 69 (5), 514-518 (2013).</li><li>Reilly, J. N., Cogger, V. C., Le Couteur, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Old+age+is+associated+with+ultrastructural+changes+in+isolated+rat+liver+sinusoidal+endothelial+cells.">Old age is associated with ultrastructural changes in isolated rat liver sinusoidal endothelial cells.</a> J Electron Microsc. 59 (1), 65-69 (2010).</li><li>Le Couteur, D. G., Rattan, A. E., Le Couteur, D. G., de Cabo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Calorie Restriction, Aging and Longevity. , 191-216 (2010).</li><li>McLean, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+pseudocapillarization+of+the+human+liver.">Age-related pseudocapillarization of the human liver.</a> J Pathol. 200 (1), 112-117 (2003).</li><li>Le Couteur, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudocapillarization+and+associated+energy+limitation+in+the+aged+rat+liver.">Pseudocapillarization and associated energy limitation in the aged rat liver.</a> Hepatology. 33 (3), 537-543 (2001).</li><li>Cogger, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+acute+oxidative+stress+on+the+ultrastructure+of+the+perfused+rat+liver.">The effect of acute oxidative stress on the ultrastructure of the perfused rat liver.</a> Pharmacol Toxicol. 89 (6), 306-311 (2001).</li><li>Horn, T., Christoffersen, P., Henriksen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcoholic+liver+injury:+defenestration+in+noncirrhotic+livers-a+scanning+electron+microscopic+study.">Alcoholic liver injury: defenestration in noncirrhotic livers-a scanning electron microscopic study.</a> Hepatology. 7 (1), 77-82 (1987).</li><li>Jamieson, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+liver+sinusoidal+endothelium+in+a+baboon+model+of+type+1+diabetes.">Alterations in liver sinusoidal endothelium in a baboon model of type 1 diabetes.</a> Diabetologia. 50 (9), 1969-1976 (2007).</li><li>Fraser, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+perfusion+pressure+damages+the+sieving+ability+of+sinusoidal+endothelium+in+rat+livers.">High perfusion pressure damages the sieving ability of sinusoidal endothelium in rat livers.</a> Br J Exp Pathol. 61 (2), 222-228 (1980).</li><li>Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+electron+microscopic+study+of+the+fenestrated+endothelial+lining+of+rat+liver+sinusoids.">An electron microscopic study of the fenestrated endothelial lining of rat liver sinusoids.</a> J Ultrastruct Res. 31 (1), 125-150 (1970).</li><li>Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ultrastructural+characterization+of+the+endothelial+cell+in+the+rat+liver+sinusoid+under+normal+and+various+experimental+conditions,+as+a+contribution+to+the+distinction+between+endothelial+and+Kupffer+cells.">An ultrastructural characterization of the endothelial cell in the rat liver sinusoid under normal and various experimental conditions, as a contribution to the distinction between endothelial and Kupffer cells.</a> J Ultrastruct Res. 38 (5), 528-562 (1972).</li><li>Fahimi, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+and+immersion+fixation+of+rat+liver+with+glutaraldehyde.">Perfusion and immersion fixation of rat liver with glutaraldehyde.</a> Lab Invest. 16 (5), 736-750 (1967).</li><li>Wisse, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+methods+for+electron+microscopy+of+human+and+other+liver.">Fixation methods for electron microscopy of human and other liver.</a> World J Gastroenterol. 16 (23), 2851-2866 (2010).</li><li>Vreuls, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jet-fixation:+a+novel+method+to+improve+microscopy+of+human+liver+needle+biopsies.">Jet-fixation: a novel method to improve microscopy of human liver needle biopsies.</a> Hepatology. 59 (2), 737-739 (2014).</li><li>Furrer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+reverts+age-related+capillarization+and+failure+of+regeneration+in+the+liver+through+a+VEGF-dependent+pathway.">Serotonin reverts age-related capillarization and failure of regeneration in the liver through a VEGF-dependent pathway.</a> Proc Natl Acad Sci U. S. A. 108 (7), 2945-2950 (2011).</li><li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OxLDL+induced+injury+and+defenestration+of+HLSECs+via+LOX-1.">OxLDL induced injury and defenestration of HLSECs via LOX-1.</a> J Mol Endocrinol. , (2014).</li><li>May, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+model+for+conditional+induction+and+rescue+of+portal+hypertension+reveals+a+role+of+VEGF-mediated+regulation+of+sinusoidal+fenestrations.">A transgenic model for conditional induction and rescue of portal hypertension reveals a role of VEGF-mediated regulation of sinusoidal fenestrations.</a> PLoS One. 6 (7), e21478 (2011).</li><li>Funyu, J., Mochida, S., Inao, M., Matsui, A., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+can+act+as+vascular+permeability+factor+in+the+hepatic+sinusoids+through+upregulation+of+porosity+of+endothelial+cells.">VEGF can act as vascular permeability factor in the hepatic sinusoids through upregulation of porosity of endothelial cells.</a> Biochem Biophys Res Commun. 280 (2), 481-485 (2001).</li><li>Venkatraman, L., Tucker-Kellogg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CD47-binding+peptide+of+thrombospondin-1+induces+defenestration+of+liver+sinusoidal+endothelial+cells.">The CD47-binding peptide of thrombospondin-1 induces defenestration of liver sinusoidal endothelial cells.</a> Liver Int. 33 (9), 1386-1397 (2013).</li><li>Wack, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinusoidal+ultrastructure+evaluated+during+the+revascularization+of+regenerating+rat+liver.">Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver.</a> Hepatology. 33 (9), 363-378 (2001).</li><li>Morsiani, E., Mazzoni, M., Aleotti, A., Gorini, P., Ricci, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+sinusoidal+wall+permeability+and+liver+fatty+change+after+two-thirds+hepatectomy:+An+ultrastructural+study+in+the+rat.">Increased sinusoidal wall permeability and liver fatty change after two-thirds hepatectomy: An ultrastructural study in the rat.</a> Hepatology. 21 (2), 539-544 (1995).</li><li>Straub, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenic-stimulated+liver+sinusoidal+capillarization+in+mice+requires+NADPH+oxidase&#8211;generated+superoxide.">Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase&#8211;generated superoxide.</a> J Clin Invest. 118 (12), 3980-3989 (2008).</li><li>Barber&#225;-Guillem, E., Arrue, J. M., Ballesteros, J., Vidal-Vanaclocha, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+in+endothelial+cells+of+developing+rat+liver+in+the+transition+from+fetal+to+postnatal+life.">Structural changes in endothelial cells of developing rat liver in the transition from fetal to postnatal life.</a> JJ Ultrastruct Mol Struct Res. 97 (1-3), 197-206 (1986).</li><li>Jamieson, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caloric+restriction+reduces+age-related+pseudocapillarization+of+the+hepatic+sinusoid.">Caloric restriction reduces age-related pseudocapillarization of the hepatic sinusoid.</a> Exp Gerontol. 42 (4), 374-378 (2007).</li><li>Cogger, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperlipidemia+and+surfactants:+the+liver+sieve+is+a+link.">Hyperlipidemia and surfactants: the liver sieve is a link.</a> Atherosclerosis. 189 (2), 273-281 (2006).</li><li>Cogger, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+oxidative+stress+on+the+liver+sieve.">The effects of oxidative stress on the liver sieve.</a> J Hepatol. 41 (3), 370-376 (2004).</li><li>Cogger, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structured+illumination+microscopy+of+liver+sinusoidal+endothelial+cell+fenestrations.">Three-dimensional structured illumination microscopy of liver sinusoidal endothelial cell fenestrations.</a> J Struct Biol. 171 (3), 382-388 (2010).</li><li>Braet, F., de Zanger, R., Seynaeve, C., Baekeland, M., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+atomic+force+microscopy+study+on+living+skin+fibroblasts+and+liver+endothelial+cells.">A comparative atomic force microscopy study on living skin fibroblasts and liver endothelial cells.</a> J Electron Microsc. 50 (4), 283-290 (2001).</li><li>Monkemoller, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+fenestrations+in+liver+sinusoidal+endothelial+cells+by+optical+localization+microscopy.">Imaging fenestrations in liver sinusoidal endothelial cells by optical localization microscopy.</a> Phys Chem Chem Phys. 16 (24), 12576-12581 (2014).</li></ol>

The authors have nothing to disclose.

The ability to accurately and reproducibly measure the status of the liver sinusoidal endothelium is an important step in understanding the biology of these highly specialized cells. Newer techniques such as structured illumination microscopy32, atomic force microscopy33&#160;and d-STORM (direct Stochastic Optical Reconstrucion Microscopy) 34 will impart important information regarding the morphology of these cells in vitro but SEM remains the primary methodology to visualize and...

The liver sinusoidal endothelial cells (LSECs) are highly differentiated endothelial cells that line the wall of the hepatic sinusoid. LSECs are perforated with fenestrations that are non-diaphragmed, transcellular pores 50-250 nm in diameter. Up to 20% of the surface of LSECs is covered by fenestrations, which are usually in groups of tens to hundreds called sieve plates1-3 (Figure 1). Fenestrations allow transfer of plasma and nanosubstrates between blood and hepatocytes, creating a highly efficient ultrafiltration system. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease. For example, fenestrations are larger in the fasted than in the fed state4; 2-di-iodoamphetamine increases fenestration number;5,6 and a reduction in size and number of fenestrations per cell occurs in ageing and many disease states7-13. Accurate measurement of the size and number of fenestrations is important for understanding how LSEC morphology is influenced by various disease, toxic, and physiological states; their impact on liver function; and for developing fenestration-modulating therapeutic interventions1.
The study of fenestrations is difficult. The diameter of fenestrations lies below the resolution of conventional light microscopy, so previously only observation using electron microscopy both in intact liver tissue or cultured LSECs has been possible. The scanning electron microscope (SEM) has most frequently been used to study fenestration size, frequency and porosity (the percentage of LSEC membrane that is perforated by fenestrations) because SEM allows for the observation of large areas of the endothelial surface and measurement of thousands, if not tens of thousands of fenestrations. Despite its utility, the results which are reported from SEM-based studies for LSEC parameters such as fenestration size, number, frequency and porosity vary widely in the literature (Table 1).
Fenestrations and sieve plates are fragile structures that contract, break, dilate or coalesce during specimen preparation, thus careful processing is needed to preserve their integrity. Elevated perfusion pressure14; incorrect osmolarity of the fixative and buffers15; inadequate fixation or fixation time; and speed of post-fixation dehydration and drying are all areas of processing for SEM that may produce artefacts that interfere with preservation of ultrastructure (Figure 2). Loss of fenestrations (&#8216;defenestration&#8217;) and fenestration shrinkage can occur as a result of poor fixation, resulting in reduced fenestration diameter and cell porosity. Methods to improve the preservation of specimens for SEM analysis have been described previously 15-17 and will be discussed here with additional tips on how to improve specimen preservation. The main goals of the specimen preservation are to remove blood from the sinusoids so that the surface of the LSEC can be visualized and to avoid LSEC damage from either high pressure or delayed fixation. Whole liver perfusion of fixative via the portal vein is the preferred method for liver fixation. As described in detail elsewhere16,18 perfusion must be undertaken at low pressure (eg 10 cm of H20) to avoid pressure-related perfusion artefacts and damage to the LSEC, typically manifested as large gaps within in the cell membrane. However, reasonable fixation can often be obtained using needle perfusion of liver biopsies from humans and animals, as described in detail elsewhere19. This technique involves directly injecting fixative into the tissue until blood is flushed out of the sample and the tissue is firm and fixed.&#160;Fixation of samples for electron microscopy needs to be performed as quickly as possible following cessation of blood flow to prevent ultrastructural changes occurring as a result of the livers extremely rapid autolytic processes.
We also present a method of image analysis that minimizes the inclusion of artefacts, and standardizes the measurement of fenestrations. Variation in the selection of sinusoids for micrographs, image analysis of artefacts, and measurement of cell area for porosity and fenestration frequency have led to major discrepancies in published results. A standardized approach for evaluation and measurement of fenestrations and the minimum requirements for data presentation have not been clearly addressed in the literature previously4,10,20-31.

<table><tbody><tr><td>25% EM grade Glutaraldehyde</td><td>ProSciTech</td><td>C001</td><td>Store stock at -20 C until needed, avoid refreeze</td></tr><tr><td>Paraformaldehyde powder</td><td>Sigma Aldrich</td><td>158127</td><td>Always prepare Paraformaldehyde fresh</td></tr><tr><td>Sodium Cacodylate powder</td><td>Sigma Aldrich</td><td>C0250</td><td>Prepare 0.2 M stock, pH 7.4 by dissolving powder in dH&lt;sub&gt;2&lt;/sub&gt;O, used mostly at 0.1 M by preparing 1:2 dilution</td></tr><tr><td>Calcium Chloride</td><td>Sigma Aldrich</td><td>C1016</td><td>Prepare 1 M CaCl2by dissolving powder in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Osmium tretroxide</td><td>ProSciTech</td><td>C011</td><td>Wash ampoules in weak acid prior to use to avoid contamination. Prepare 2 % stock in glass bottle</td></tr><tr><td>Ethanol- Absolute</td><td>Sigma Aldrich</td><td>459836&amp;nbsp;</td><td>100 % Ethanol must be high grade and stored with Molecular Sieve</td></tr><tr><td>Other grades of Ethanol</td><td>Labtech</td><td>EL5</td><td>Prepare graded Ethanols with dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Hexamethyldisilazane</td><td>Sigma Aldrich</td><td>52619&amp;nbsp;</td><td>Allow to reach room temperature before use</td></tr><tr><td>Cannulas</td><td>Terumo</td><td>TSROX1832C, TSROX2225C, TSROX2419C</td><td>18 G is suitable for most rats, 22 G is suitable for most mice, but it is good to have a few 24 G on hand in case of very small mice</td></tr><tr><td>Conductive Carbon tape</td><td>ProSciTech</td><td>IA0201</td><td></td></tr><tr><td>Carbon Paint</td><td>ProSciTech</td><td>I003</td><td></td></tr><tr><td>Ketamine</td><td></td><td></td><td>Must be obtained under license</td></tr><tr><td>Xylazine</td><td></td><td></td><td>Must be obtained under license</td></tr><tr><td>Molecular Sieve</td><td>Sigma Aldrich</td><td>208647</td><td>Removes water from the 100 % Ethanol</td></tr></tbody></table>

a standardized method for the analysis of liver sinusoidal endothelial cells and their fenestrations by scanning electron microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

Initial visualization at low magnification by scanning electron microscopy reveals a flat surface of the liver specimen with an exposed area large enough to observe many large liver vessels and sinusoids (Figure 1A). Ensuring correct liver block placement on the mounting stub is essential for obtaining clear images of the sinusoids and the Glisson&#8217;s capsule of the liver should be avoided for this reason (Figure 1B). Increasing the magnification allows closer inspection of the dense...

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A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

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Research

JoVE Journal

Biology

14.4K Views.  University of Sydney and Concord Hospital. The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

Video: A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Biochemistry

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

Cancer Research

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Immunology and Infection

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...

Medicine

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of the intact endothelium will provide valuable information for understanding the cellular and molecular events in the dysfunctional endothelial cells. Here, we describe a modified en face immunofluorescence staining technique which enables scientists to obtain clear images of the intact endothelial surface and analyze the molecule expression patterns in situ. The method is simple and reliable for observing the entire endothelial monolayer at different sites of the aorta. This technique may be a promising tool for understanding the pathophysiology of atherosclerosis, especially at an early stage.

Here, we present a protocol for immunofluorescence staining to observe the endothelial cells of the mouse aorta directly. This technique is useful when studying the cellular and molecular phenotype of endothelial cells in different flow patterns and in the development of atherosclerosis.

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

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Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells