Name: histological-based stainings using free-floating tissue sections
Uploaded: 2020-08-25T14:30:00
Description: Watch this Scientific Journal Video about Histological-Based Stainings Using Free-Floating Tissue Sections at JoVE.com

We would like to acknowledge the National Institute on Aging (K99/R00 AG055683 to JMR), the George and Anne Ryan Institute for Neuroscience (EP, GC, JMR), the College of Pharmacy at the University of Rhode Island (EP, GC, JMR), and Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse (JMR). We thank doctoral student Rebecca Senft, training with Professor Susan Dymecki, Department of Genetics, Harvard Medical School, for introducing us to the free-floating method. Some images used in Figure 1 were obtained from &#34;free to use, share, or modify, even commercially&#8221; sources: mouse and microcentrifuge tube (Pixabay), mouse brain (Jonas T&#246;le, Wikimedia Commons), cryostat and mouse brain section (DataBase Center for Life Science, Wikimedia Commons), glass container (OpenClipart, FreeSvg.org), and microscope (Theresa Knott, Open Clip Art Library).

1. Tissue preparation for cryosectioning
<ol>
	<li>Embed fixed tissues in an appropriate embedding mold (see Table of Materials) to create a specimen block using an appropriate specimen matrix (see Table of Materials) and freeze on dry ice. Store specimen blocks at -80 &#176;C until ready to section. 
	NOTE: Fixed tissues are typically prepared by perfusing adult (approx. 2.5 &#8211; 30 months old) male or female rodents (mouse or rat)34, in accordance with...

<ol>
	<li>Childs, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathobiology+of+Human+Disease.+Part+IV:+Techniques+in+Experimental+Pathology.">Pathobiology of Human Disease. Part IV: Techniques in Experimental Pathology.</a> Journal of Experimental Medicine. 381923, 3775-3796 (2014).</li><li>Coons, A. H., Creech, H. J., Jones, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+Properties+of+an+Antibody+Containing+a+Fluorescent+Group.">Immunological Properties of an Antibody Containing a Fluorescent Group.</a> Experimental Biology and Medicine. 47 (2), 200-202 (1941).</li><li>Goldman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies:+indispensable+tools+for+biomedical+research.">Antibodies: indispensable tools for biomedical research.</a> Trends in Biochemical Sciences. 25 (12), 593-595 (2000).</li><li>Taylor, S. N., Harlow, E. D., Lane, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Antibodies:+A+Laboratory+Manual.">Using Antibodies: A Laboratory Manual.</a> The Quarterly Review of Biology. 74 (3), 374 (1999).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proceedings of the NationalAcademy of Sciences. 76 (9), 4350-4354 (1979).</li><li>Hermersd&#246;rfer, H., Beesley, J. 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> Immunocytochemistry. A Practical Approach. 38 (3), 348-348 (1994).</li><li>Schacht, V., Kern, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basics+of+Immunohistochemistry.">Basics of Immunohistochemistry.</a> Journal of Investigative Dermatology. 135 (3), 1-4 (2015).</li><li>Coons, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Review+of+Cytology.">International Review of Cytology.</a> International Review of Cytology. 5, 1-23 (1956).</li><li>Mepham, B. L., Britten, K. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunostaining+Methods+for+Frozen+and+Paraffin+Sections.">Immunostaining Methods for Frozen and Paraffin Sections.</a> Lymphoproliferative Diseases. , 187-211 (1990).</li><li>Duraiyan, J., Govindarajan, R., Kaliyappan, K., Palanisamy, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+immunohistochemistry.">Applications of immunohistochemistry.</a> Journal of Pharmacy &amp; Bioallied Sciences. 4, 307-309 (2012).</li><li>Li, A., Yang, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+Molecular+Biology.">Methods in Molecular Biology.</a> Methods in Molecular Biology. 2108, 43-55 (2020).</li><li>H&#246;kfelt, T., Fuxe, K., Goldstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Immunohistochemistry+to+Studies+on+Monoamine+Cell+Systems+with+Special+Reference+To+Nervous+Tissues.">Applications of Immunohistochemistry to Studies on Monoamine Cell Systems with Special Reference To Nervous Tissues.</a> Annals of the New York Academy of Sciences. 254, 407-432 (1975).</li><li>Okaty, B. W., 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+single-cell+transcriptomic+and+anatomic+atlas+of+mouse+dorsal+raphe+Pet1+neurons.">A single-cell transcriptomic and anatomic atlas of mouse dorsal raphe Pet1 neurons.</a> bioRxiv. , (2020).</li><li>Koenig, H., Groat, R. A., Windle, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Physiological+Approach+to+Perfusion-Flxation+of+Tissues+with+Formalin.">A Physiological Approach to Perfusion-Flxation of Tissues with Formalin.</a> Stain Technology. 20 (1), 13-22 (1945).</li><li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fixatives+for+immunostaining.">Cell fixatives for immunostaining.</a> Methods in Molecular Biology. 588, 55-61 (2010).</li><li>van der Loos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+focus+on+fixation.">A focus on fixation.</a> Biotechnic &amp; Histochemistry. 82 (3), 141-154 (2007).</li><li>Canene-Adams, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+formalin-fixed+paraffin-embedded+tissue+for+immunohistochemistry.">Preparation of formalin-fixed paraffin-embedded tissue for immunohistochemistry.</a> Methods in Enzymology. 533, 225-233 (2013).</li><li>Bachman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+on+freely+floating+fixed+tissue+sections.">Immunohistochemistry on freely floating fixed tissue sections.</a> Methods in Enzymology. 533, 207-215 (2013).</li><li>Sundquist, S. J., Nisenbaum, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Fos:+rapid+protocols+for+single-+and+double-labeling+c-Fos+immunohistochemistry+in+fresh+frozen+brain+sections.">Fast Fos: rapid protocols for single- and double-labeling c-Fos immunohistochemistry in fresh frozen brain sections.</a> Journal of Neuroscience Methods. 141 (1), 9-20 (2005).</li><li>Bertheau, P., 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=Variability+of+immunohistochemical+reactivity+on+stored+paraffin+slides.">Variability of immunohistochemical reactivity on stored paraffin slides.</a> Journal of Clinical Pathology. 51 (5), 370-374 (1998).</li><li>Blind, 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=Antigenicity+testing+by+immunohistochemistry+after+tissue+oxidation.">Antigenicity testing by immunohistochemistry after tissue oxidation.</a> Journal ofClinical Pathology. 61 (1), 79-83 (2007).</li><li>Desmet, V. J., Krstulovi&#263;, B., Damme, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+study+of+rat+liver+in+alpha-naphthyl+isothiocyanate+(ANIT)+induced+cholestasis.">Histochemical study of rat liver in alpha-naphthyl isothiocyanate (ANIT) induced cholestasis.</a> The American Journal of Pathology. 52 (2), 401-421 (1968).</li><li>Bulmer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Histochemistry+of+Ovarian+Macrophages+in+the+Rat.">The Histochemistry of Ovarian Macrophages in the Rat.</a> Journal of Anatomy. 98, 313-319 (1964).</li><li>L&#246;nnerholm, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+Demonstration+of+Carbonic+Anhydrase+Activity+in+the+Human+Kidney.">Histochemical Demonstration of Carbonic Anhydrase Activity in the Human Kidney.</a> Acta Physiologica Scandinavica. 88 (4), 455-468 (1973).</li><li>Ronnekleiv, O. K., Naylor, B. R., Bond, C. T., Adelman, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+Immunohistochemistry+for+Gonadotropin-Releasing+Hormone+(GnRH)+and+Pro-GnRH,+and+in+Situ+Hybridization+for+GnRH+Messenger+Ribonucleic+Acid+in+Rat+Brain.">Combined Immunohistochemistry for Gonadotropin-Releasing Hormone (GnRH) and Pro-GnRH, and in Situ Hybridization for GnRH Messenger Ribonucleic Acid in Rat Brain.</a> Molecular Endocrinology. 3 (2), 363-371 (1989).</li><li>Nadelhaft, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sessile+drop+method+for+immunohistochemical+processing+of+unmounted+sections+of+nervous+tissue.">The sessile drop method for immunohistochemical processing of unmounted sections of nervous tissue.</a> Journal of Histochemistry &amp; Cytochemistry. 32 (12), 1344-1346 (1984).</li><li>Wainer, B. H., Rye, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+horseradish+peroxidase+tracing+combined+with+localization+of+choline+acetyltransferase+immunoreactivity.">Retrograde horseradish peroxidase tracing combined with localization of choline acetyltransferase immunoreactivity.</a> Journal of Histochemistry &amp; Cytochemistry. 32 (4), 439-443 (1984).</li><li>Piekut, D. T., Casey, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetration+of+immunoreagents+in+Vibratome-sectioned+brain:+a+light+and+electron+microscopic+study.">Penetration of immunoreagents in Vibratome-sectioned brain: a light and electron microscopic study.</a> Journal of Histochemistry &amp; Cytochemistry. 31 (5), 669-674 (1983).</li><li>Cowan, R. L., Wilson, C. J., Emson, P. C., Heizmann, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin-containing+gabaergic+interneurons+in+the+rat+neostriatum.">Parvalbumin-containing gabaergic interneurons in the rat neostriatum.</a> The Journal of Comparative Neurology. 302 (2), 197-205 (1990).</li><li>Kjell, 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=Delayed+Imatinib+Treatment+for+Acute+Spinal+Cord+Injury:+Functional+Recovery+and+Serum+Biomarkers.">Delayed Imatinib Treatment for Acute Spinal Cord Injury: Functional Recovery and Serum Biomarkers.</a> Journal of Neurotrauma. 32 (21), 1645-1657 (2015).</li><li>Sterky, F. H., Lee, S., Wibom, R., Olson, L., Larsson, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+mitochondrial+transport+and+Parkin-independent+degeneration+of+respiratory+chain-deficient+dopamine+neurons+in+vivo.">Impaired mitochondrial transport and Parkin-independent degeneration of respiratory chain-deficient dopamine neurons in vivo.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (31), 12937-12942 (2011).</li><li>Watson, R. E., Wiegand, S. J., Clough, R. W., Hoffman, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+cryoprotectant+to+maintain+long-term+peptide+immunoreactivity+and+tissue+morphology.">Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology.</a> Peptides. 7 (1), 155-159 (1986).</li><li>Estrada, L. I., 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=Evaluation+of+Long-Term+Cryostorage+of+Brain+Tissue+Sections+for+Quantitative+Histochemistry.">Evaluation of Long-Term Cryostorage of Brain Tissue Sections for Quantitative Histochemistry.</a> The Journal of Histochemistry and Cytochemistry. 65 (3), 153-171 (2017).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</li><li>Griffiths, G., McDowall, A., Back, R., Dubochet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+preparation+of+cryosections+for+immunocytochemistry.">On the preparation of cryosections for immunocytochemistry.</a> Journal of Ultrastructure Research. 89 (1), 65-78 (1984).</li><li>Capelozzi, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Papel+da+imuno-histoqu&#237;mica+no+diagn&#243;stico+do+c&#226;ncer+de+pulm&#227;o.">Papel da imuno-histoqu&#237;mica no diagn&#243;stico do c&#226;ncer de pulm&#227;o.</a> Jornal Brasileiro de Pneumologia. 35 (4), 375-382 (2009).</li><li>Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunocytochemistry. , 65-77 (2009).</li><li>Grabinski, T. M., Kneynsberg, A., Manfredsson, F. P., Kanaan, N. 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+method+for+combining+RNAscope+in+situ+hybridization+with+immunohistochemistry+in+thick+free-floating+brain+sections+and+primary+neuronal+cultures.">A method for combining RNAscope in situ hybridization with immunohistochemistry in thick free-floating brain sections and primary neuronal cultures.</a> PloS one. 10 (3), 0120120 (2015).</li><li>Warr, W. B., de Olmos, J. 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Immunohistochemistry (IHC) is a versatile technique that has become crucial in identifying protein expression and localization within tissue sections. This assay is used throughout the scientific community to further understand characteristics of tissue across stages of normal function to disease-states. IHC is employed across a variety of fields from clinical diagnosis of diseases such as cancer to initial discoveries in preclinical research10,36.
<p class="...

Immunostaining is a popular research practice that began 130 years ago with the discovery of serum antibodies in 1890 by Von Behring1. During the early 20th century, dyes were attached to antigens and later to antibodies as a way to quantify and visualize reactions1, and in 1941 Albert Coons developed the first fluorescent antibody labels, a discovery that revolutionized light microscopy2,3. The term &#8220;immunostaining&#8221; encompasses many techniques that have been developed using this principle, including Western blot, flow cytometry, ELISA, immunocytochemistry, and immunohistochemistry3,4. Western blot detects the presence of specific proteins from tissue or cell extracts5. Proteins are separated by size using gel electrophoresis, transferred to a membrane, and probed using antibodies. This technique indicates the presence of protein and how much protein is present; however, it does not reveal any information on the localization of the protein within cells or tissues. Another method, immunocytochemistry (ICC), labels proteins within cells, typically cells cultivated in vitro. ICC shows both protein expression and localization within cellular compartments6. To detect and visualize a specific protein at the tissue level, immunohistochemistry (IHC) is utilized.
IHC is a method that researchers use to target specific antigens within tissue, taking advantage of chemical properties of the immune system7,8. By generating specific primary and secondary antibodies linked to either an enzyme or a fluorescent dye, antigens of interest can be labelled and revealed in most tissues (as reviewed in Mepham and Britten)9. The term &#8220;immunohistochemistry&#34; by itself does not specify the labeling method that is used to reveal the antigen of interest; thus, this terminology is often combined with the detection technique to clearly delineate the labeling method: chromogenic immunohistochemistry (CIH) to indicate when the secondary antibody is conjugated to an enzyme, such as peroxidase; or fluorescent IHC to indicate when the secondary antibody is conjugated to a fluorophore, such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine (TRITC). The selectivity of IHC allows clinicians and researchers to visualize protein expression and distribution throughout tissues, across various states of health and disease10. In the clinical realm, IHC is commonly used to diagnose cancer, as well as to determine differences in various types of cancer. IHC has also been used to confirm different types of microbial infections within the body, such as Hepatitis B or C11. In biomedical research, IHC is often used to map protein expression in tissues and is important in identifying abnormal proteins seen in disease states. For example, neurodegeneration is often accompanied by accumulation of abnormal proteins in the brain, such as &#913;&#946; plaques and neurofibrillary tangles in Alzheimer&#8217;s disease. Animal models are often then developed to mimic these pathological states, and IHC is one method that researchers use to locate and quantify the proteins of interest10,12,13. In turn, we can learn more about the causes of these diseases, and the complications that arise with them.
There are many steps involved in performing IHC. First, the tissue of interest is collected and prepared for staining. Arguably most researchers prepare fixed tissue samples, with perfusion of the fixative via the circulatory system being optimal as it preserves morphology14,15. Post-fixation of tissue samples may also be used but may yield less than ideal results16. Crosslinking fixatives, such as formaldehyde, act by creating chemical bonds between proteins in the tissue17. Fixed tissue is then sliced into very thin layers or sections using a microtome, with many researchers preferring to collect frozen sections using a cryostat. From there the tissue is collected and either mounted directly onto a microscope slide (slide-mounted method), or suspended in a solution (free-floating method), such as phosphate buffered saline (PBS)18. The method of collection used is predetermined based on the needs of the researcher, with each of these two methods presenting its own advantages and disadvantages.
The slide-mounted method is by far the most commonly used, with an important benefit being that very thin sections (10-14 &#956;m) can be prepared, which is important, for example, to investigate protein-protein interactions. There is also minimal handling of the specimen, which decreases potential damage to the structural integrity of the tissue19. Researchers often use this technique with fresh frozen tissue (tissue that is immediately frozen using dry ice, isopentane, etc.), which is very delicate as compared to fixed tissue and much care to prevent thawing of the sample needs to be taken. Another advantage of using slide-mounted sections is that large volumes of solutions for staining are usually not required4. Thus, researchers can use a smaller amount of expensive antibodies or other chemicals to complete the stain. Additionally, it is possible to mount sections from several different experimental groups on the same slide, which can be advantageous, especially during image acquisition. On the other hand, there are some disadvantages of using slide-mounted sections, most notably that the tissue section is adhered to the slide thus restricting antibody penetration to one side of the section, which limits the section thickness and the 3D representation of the tissue. It can also happen that during washings, the edges of the tissue and entire sections may detach from the slide, rendering useless the whole experiment. Moreover, IHC usually has to be performed relatively quickly when using the slide-mounted approach to avoid degradation of the antigen epitope20,21 with unprocessed slides typically stored at -20 or -80 &#176;C, often coverslipped and stored horizontally or in slide boxes, resulting in a relatively large storage footprint. Lastly, the slide-mounted technique can also be time consuming if researchers must handle large numbers of slides to process large numbers of tissue sections.
Due to some of these challenges using the slide-mounted method, a modification called the free-floating method has become a popular alternative. This technique came into the literature in the 1960-70s22,23,24, gaining popularity in the 1980s25,26,27,28,29, and is now a well-established method that involves performing the stain on the collected sections in suspension rather than adhered to a slide12,30,31. The free-floating method is not recommended when tissue sections are less than 20 &#956;m; however, in our experience it is the approach of choice when thicker (40-50 &#956;m) sections are to be stained. One distinct benefit is that antibodies can penetrate free-floating sections from all angles and generate less background staining due to more effective washing, all resulting in better signaling when imaging. Additionally, the sections are mounted onto the slides after processing, thus eliminating the possibility of tissue detachment as well as decreasing the time occupying the cryostat. The free-floating method can also be much less labor intensive, especially for large-scale biomedical studies. For instance, it is possible to stain many (18-40) sections from the same sample together in the same well, which saves time in performing both the wash and antibody incubation steps. Moreover, since a larger number (12-16) of sections can be mounted per slide using this approach, it is often more convenient and quicker for the researcher to view and image sections. Notably, during the mounting of tissue slices on the slides, sections can be attached and detached until the desired orientation is obtained. Researchers also often use slightly lower concentrations of antibodies using the free-floating method, and since the incubations are performed in microcentrifuge tubes, the antibodies can be easily collected and preserved with sodium azide for reusage (see Step 5.1). Another advantage is that the sections can be directly stored at -80 &#176;C in small microcentrifuge tubes with cryoprotectant solution32, thereby minimizing storage space and maximizing longevity of the samples33. A down-side of using this technique is that the sections are handled a lot, and thus are apt to damage. This, however, can be mitigated by using low shaking and rotating speeds as well as properly training researchers how to transfer the samples and mount the sections onto the slides.
Taken together, IHC is an established, essential tool for visualizing and localizing protein expression in both the clinical and biomedical research fields. Free-floating IHC is an efficient, flexible, as well as economic method, especially when performing large-scale histological studies. Here, we present a reliable free-floating fluorescent IHC protocol for the scientific community that can be adapted accordingly for chromogenic IHC and other stainings such as hematoxylin and eosin or cresyl violet staining.

<table>
	<tbody>
		<tr>
			<td>12-well plates</td>
			<td>Corning</td>
			<td>3513</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear nail polish</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding molds</td>
			<td>Thermo Scientific</td>
			<td>1841</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution</td>
			<td>Fisher Scientific</td>
			<td>SF98-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum, heat inactivated</td>
			<td>Gibco</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide boxes</td>
			<td>Electron Microscopy Services</td>
			<td>71370</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary antibody</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Rectangular Coverslips</td>
			<td>VWR</td>
			<td>48393-081</td>
			<td>24 x 50 mm</td>
		</tr>
		<tr>
			<td>Rectangular staining dish</td>
			<td>Electron Microscopy Services</td>
			<td>70312</td>
			<td></td>
		</tr>
		<tr>
			<td>Round artist paintbrush #2</td>
			<td>Princeton Select Series</td>
			<td>3750R</td>
			<td>Brand not important</td>
		</tr>
		<tr>
			<td>Secondary antibody</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen matrix for embedding</td>
			<td>OCT Tissue-Tek, Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Stain tray &#8211; slide staining system</td>
			<td>Electron Microscopy Services</td>
			<td>71396-B</td>
			<td>Use dark lid</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
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			<td>Superfrost Plus Micro Slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
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			<td>TBS</td>
			<td>User preference</td>
			<td>N/A</td>
			<td></td>
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			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
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		<tr>
			<td>Vectashield antifade mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Non-hardening</td>
		</tr>
		<tr>
			<td>Well inserts for 12-well plates</td>
			<td>Corning Netwells</td>
			<td>3477</td>
			<td></td>
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			<td>Well inserts for 6-well plates</td>
			<td>Corning Netwells</td>
			<td>3479</td>
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			<td>Whatman filter paper</td>
			<td>Millapore-Sigma</td>
			<td>WHA1440042</td>
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histological-based stainings using free-floating tissue sections

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are widely used to handle the tissue sections during the staining procedure, one approach consists of mounting the sections directly on glass slides, while a second approach, the free-floating, allows for fixed sections to be maintained and stained while suspended in solution. Although slide-mounted and free-floating approaches may yield similar results, the free-floating technique allows for better antibody penetration and thus should be the method of choice when thicker sections are to be used for 3D reconstruction of the tissues, for example when the focus of the experiment is to gain information on dendritic and axonal projections in brain regions. In addition, since the sections are kept in solution, a single aliquot can easily accommodate 30 to 40 sections, handling of which is less laborious, particularly in large-scale biomedical studies. Here, we illustrate how to apply the free-floating method to fluorescent immunohistochemistry staining, with a major focus on brain sections. We will also discuss how the free-floating technique can easily be modified to fit the individual needs of researchers and adapted to other tissues as well as other histochemical-based stainings, such as hematoxylin and eosin and cresyl violet, as long as tissue samples are properly fixed, typically with paraformaldehyde or formalin.

The overall scheme of the using the free-floating method to perform a fluorescent immunohistochemical assay is illustrated in Figure 1. Representative example of fluorescent IHC using the free-floating method in mouse brain examining glial fibrillary acidic protein (GFAP) expression is shown in Figure 2 at both lower and higher magnification to illustrate the overall quality of the staining. This approach is also appropriate for revealing low-expressing proteins...

Watch this Scientific Journal Video about Histological-Based Stainings Using Free-Floating Tissue Sections at JoVE.com

Histological-Based Stainings Using Free-Floating Tissue Sections

The free-floating technique allows researchers to perform histological-based stainings including immunohistochemistry on fixed tissue sections to visualize biological structures, cell type, and protein expression and localization. This is an efficient and reliable histochemical technique that can be useful for investigating a multitude of tissues, such as brain, heart, and liver.

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative ...

This free floating technique can be used to perform for lessened immunohistochemistry on mouse brain tissue samples. The free floating technique provides several advantages. The acquisition of thicker tissue sections, the performance of large scale studies, using 30 to 40 slices per aliquot and the long-term storage of samples.If you are trying this technique for the first time, we recommend you practice mounting sections that are not critical to your experiment to obtain experience with the method. At least one to two hours before sectioning, acclimatize the samples of interest in the cryostat at the appropriate tissue cutting temperature. When the tissues are ready.use the cryostat to acquire 20 to 50 micron thick sections and place the sections into individual inserts in PBS filled wells of a six or 12 well plate. When all of the sections have been acquired, cover the samples with approximately six milliliters per wash for three five minute washes before transferring each well of sections into two milliliter micro centrifuge tubes, containing one to 1.5 milliliters of storage solution per tube. Then store the samples at minus 80 degrees Celsius until staining.For tissue section staining, equilibrate the samples to room temperature for 10 to 20 minutes before decanting the sections into inserts in a new six well plate. When all of the storage solution has been drained, move the inserts into new wells, containing approximately six milliliters of TBS per well for three washes for five minutes with fresh TBS per wash on an orbital shaker at low speed at room temperature. After the last wash, add seven milliliters of freshly prepared blocking permeabilizing solution to each well for a 30 minute incubation on the shaker at room temperature.At the end of the incubation, add one milliliter of the primary antibody solution of interest to one two milliliter micro centrifuge tube per insert and transfer all of the sections in each insert into the appropriate two milliliter tube. Then place the tubes on a rotating mixer. Add about seven revolutions per minute for an overnight incubation at four degrees Celsius.The next morning, decant the sections from each tube into individual well inserts in a new six well plate and wash the sections two times for 30 seconds per wash and one time for 10 minutes in fresh TBS per wash. After the last wash, transfer the groups of sections into individual two milliliter micro centrifuge tubes, containing one milliliter of the appropriate secondary antibody solution per tube and incubate the samples for two hours at room temperature on an orbital shaker at low speed, protected from light. At the end of the incubation, decant the sections into inserts in a new six well plate and wash the samples two times for 30 seconds and one time for 15 minutes in fresh TBS per wash, protected from light.After the last wash, decant the sections into a glass rectangular histological chamber, three quarters filled with TBS and submerge a glass slide in the TBS. Using a fine paintbrush and a magnifying lamp if desired, carefully coax the sections toward the slide and gently tap a section onto the slide, making sure there are no wrinkles or folds. When all of the sections have been captured onto a slide, allow the slides to dry for about 10 to 15 minutes at room temperature, protected from light, until the sections are opaque and apply an appropriate aqueous mounting medium to the sections.Use tweezers to place a cover slip onto the medium and use a piece of filter paper to firmly press down on the cover slip to remove any excess medium. The sections can then be imaged, using an appropriate microscope or stored in a dark slide box set four degree Celsius. As observed in these representative images, fluorescent immunohistochemical analysis, using the free floating method can be performed on mouse brain tissue sections to examine glial fibrillar acidic protein expression.This approach is also appropriate for revealing low expression proteins, such as GFP, in a low expressing transgenic mouse brain sample or in non fluorescent histochemical staining protocols, such as cresyl violet. Other peripheral tissues are also amenable to use of this technique with no modifications required as observed for these low level GFP expressing liver tissue samples. The most important thing to remember with the free floating technique is to be gentle with the sections, especially during transfers and when the samples are on the shakers or rotators.In addition to a immunofluorescence staining, this method can be easily modified for other histochemical stains such as hematoxylin and eosin and cresyl violet or for chromogen immunohistochemistry.

histological-based-stainings-using-free-floating-tissue-sections

Research

JoVE Journal

Biology

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3. On the macro scale the stiffness of tissues and organs within the human body span several orders of magnitude4. Much less is known about how stiffness varies spatially within tissues, and what the scope and spatial scale of stiffness changes are in disease processes that result in tissue remodeling. To better understand how changes in matrix stiffness contribute to cellular physiology in health and disease, measurements of tissue stiffness obtained at a spatial scale relevant to resident cells are needed. This is particularly true for the lung, a highly compliant and elastic tissue in which matrix remodeling is a prominent feature in diseases such as asthma, emphysema, hypertension and fibrosis. To characterize the local mechanical environment of lung parenchyma at a spatial scale relevant to resident cells, we have developed methods to directly measure the local elastic properties of fresh murine lung tissue using atomic force microscopy (AFM) microindentation. With appropriate choice of AFM indentor, cantilever, and indentation depth, these methods allow measurements of local tissue shear modulus in parallel with phase contrast and fluorescence imaging of the region of interest. Systematic sampling of tissue strips provides maps of tissue mechanical properties that reveal local spatial variations in shear modulus. Correlations between mechanical properties and underlying anatomical and pathological features illustrate how stiffness varies with matrix deposition in fibrosis. These methods can be extended to other soft tissues and disease processes to reveal how local tissue mechanical properties vary across space and disease progression.

The stiffness of the extracellular matrix strongly influences multiple behaviors of adherent cells. Matrix stiffness varies spatially throughout a tissue, and undergoes modification in various disease conditions. Here we develop methods to characterize spatial variations in stiffness in normal and fibrotic mouse lung tissue using atomic force microscopy microindentation.

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...