We thank all the members from our lab and Ophthalmic Animal Laboratory of Zhongshan Ophthalmic Center for their technical assistance. We also thank Prof. Chunqiao Liu for experimental support. This work was supported by grants from the National Natural Science Foundation of China (NSFC: 81670872; Beijing, China), the Natural Science Foundation of Guangdong Province, China (Grant No.2019A1515011347), and High-level hospital construction project from State Key Laboratory of Ophthalmology at Zhongshan Ophthalmic Center (Grant No. 303020103; Guangzhou, Guangdong Province, China).

All procedures involving the use of mice were approved by the animal experimental ethics committee of Zhongshan Ophthalmic Center, Sun Yat-sen University, China (authorized number: 2020-082), and in accordance with the approved guidelines of Animal Care and Use Committee of Zhongshan Ophthalmic Center and the Association Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.
1. Induction of mouse OIR model
<ol>
	<li>Use mice with a lower rate of congenital malformation of the eyes, e.g., C57BL/6J mice, and mate them at a ratio of male/female = 1:2. Get the pups born on the same day and start to induce the OIR model at P7. Record the bodyweight of mouse pups strictly before modeling. 
	NOTE: Note the day of birth as P0. Record the weight of each mouse regularly. The bodyweight of newborn pups is very important during the induction of OIR as the sensitivity of mice in different states to oxygen is different. Exclude the pups more than 5 g at P7 to ensure comparable results.</li>
	<li>Provide a suitable living environment for nursing mothers and their pups, such as setting the temperature at 23 &#176;C &#177; 2 &#176;C, controlling the humidity at 40%-65%, alternating 12 h of light and 12 h of darkness every day, adding some cotton wool to the cage for nesting, ensuring adequate sterilized food and water, and keeping them in individually ventilated cages (IVC).</li>
	<li>Monitor the level of humidity and temperature inside the chamber. Control the humidity between 40% to 65% and keep the temperature at 23 &#176;C &#177; 2 &#176;C.</li>
	<li>Check the oxygen supply with oxygen sensors, maintain a constant oxygen level at 75% and control the oxygen flow rate at 0.5-0.75 L/min. Put 50 g soda lime at the bottom of the chamber to absorb excessive CO2 and maintain CO2 values below 3%22.</li>
	<li>Monitor the behaviors of nursing mothers such as nest-building behavior, biting their pups, and refusing lactation at least once a day. Eliminate nursing mothers with poor motherhood.</li>
	<li>Place the P7 pups (male and female) and their nursing mothers into an oxygen chamber in which the oxygen level is 75% for 5 days to P12. Avoid unnecessary opening of the chamber during the period of model induction. Ensure that there are extra surrogate mothers for replacement, in case the nursing mothers die due to lung injury while in hyperoxia. 
	NOTE: To ensure the comparability of the experiment, restrict the number to 6-8 pups for each mother. Pay attention to the potential problem of oxygen toxicity, which causes the death of some nursing mothers. The signs of hyperoxic lung injury in nursing mothers include, but are not limited to, fluctuating respiratory rate, decreased activity, and decreased feeding. When the above phenomenon occurs, euthanize the nursing mother with 1% pentobarbital sodium (50 mg/kg) as soon as possible. Prepare some surrogate mothers, e.g., 129S1/SvImJ for replacement and use them only if necessary. It is not recommended to replace nursing mothers as a routine, as this will lead to frequent opening of an oxygen chamber, resulting in unstable oxygen levels and maternal aggression.</li>
	<li>Bring the pups and their nursing mothers back to the room air at P12 and monitor the weight of all the pups continuously until P17. Group the pups based on the weight to ensure that each experimental group has a similar weight distribution.</li>
</ol>
2. Preparation of retinal whole mounts and immunofluorescence staining
<ol>
	<li>Record the bodyweight of the pups. Sacrifice the pups by an overdose of anesthetic (1% pentobarbital sodium 50 mg/kg) or CO2 inhalation. Other methods of euthanasia, such as cervical dislocation and bilateral thoracotomy, can be used if necessary.</li>
	<li>Use curved scissors to release the connection between eyeballs and orbital tissue. Then, put curved forceps into the posterior part of the eyeball, clamp the optic nerve, and quickly lift the eye out from the orbit. Wash the eyeballs in a pre-cooled 1x phosphate buffer saline (PBS) to remove the hair and blood from the surface of the eyeballs.</li>
	<li>Place the cleaned eyeballs in a 2 mL microcentrifuge tube filled with 4% paraformaldehyde (PFA) and incubate for 15 min at room temperature on a shaker at a speed of 12-15 revolutions per minute (rpm) (initial fixation). 
	CAUTION: Paraformaldehyde is known to be allergenic, generally toxic, and extremely cytotoxic. Follow the safety instructions strictly and avoid inhalation and skin contact.</li>
	<li>Use a culture dish and put a drop of 1x PBS into the central part and perform the following steps under a dissecting microscope and place one eyeball in this drop. Hold the eyeball with a pair of forceps and carefully puncture the cornea at the corneal limbus using a 1 mL syringe needle. Insert the tip of the scissors into this hole and cut off the cornea carefully along the cornea limbus. Be careful not to cut the retina.</li>
	<li>Remove the iris and lens with a pair of forceps. Then place the remaining eyecup in the 4% PFA and fix again for another 45 min at room temperature on a shaker at a speed of 12-15 rpm (secondary fixation).</li>
	<li>Use a culture dish and put a drop of 1x PBS into the central part. Place the fixed eyeball in this drop. Hold the eyeball with a pair of forceps. Gently separate the retina and sclera layers using two forceps. Place the tip of the scissors between the retina and sclera layers and cut the sclera toward the optic nerve. Peel the sclera off the retina and obtain the retinal cup. 
	NOTE: Hold the posterior cup by the optic nerve with forceps, then use the curved end of another forceps to press down on the sclera at the optic nerve head and gently massage out the retina in a forward sweeping motion as an alternative to release the retina.</li>
	<li>Use forceps to release the connection between radial hyaloid vessels and peripheral retina, clamp the root of the hyaloid vessels which is close to the optic nerve head, and cut the hyaloid vessels off carefully.</li>
	<li>Use a 2 mL pipette with the tip cut off to transfer the retinal cup. Place the retinal cup into one well in a 48-well plate and wash it for 3 x 5 min with 1x PBS at room temperature on a shaker at a speed of 12-15 rpm.</li>
	<li>Incubate the retinal cup in a mixed solution of 1% Triton X-100 (in PBS) and 5% normal donkey serum (in PBS) overnight at 4 &#176;C.
	<ol>
		<li>Alternatively, block and permeabilize retinas at room temperature for 1 h as an alternative. Change blocking serum according to the source of the secondary antibody.</li>
	</ol>
	</li>
	<li>If labeling the retinal vasculature using Isolectin B4, incubate the retina in a well of 48-well plate with 0.1% normal donkey serum (400 &#181;L) and IsolectinB4-594 (1:400) overnight at 4 &#176;C on a shaker at a speed of 12-15 rpm. 
	NOTE: If labeling the blood vessels with other markers, such as CD31, or labeling other cells, use specific primary antibodies to label them.</li>
	<li>Incubate the retina with 1:100-1:500 specific primary antibodies (in 400 &#181;L 0.1% normal donkey serum) at 4 &#176;C on a shaker at a speed of 12-15 rpm for 48 h. (optional)</li>
	<li>After returning to the room temperature, wash the retina with 0.1% PBST (0.1% TritonX-100 in PBS) for 3 x 20 min on a shaker at a speed of 12-15 rpm.</li>
	<li>Incubate the retina with 1:1,000 secondary antibodies (in 400 &#181;L 0.1% normal donkey serum) overnight at 4 &#176;C on a shaker at a speed of 12-15 rpm. (optional)
	<ol>
		<li>Alternatively, incubate the retina with high-affinity secondary antibodies at room temperature for 1 h.</li>
	</ol>
	</li>
	<li>Incubate the retina with DAPI (1:1,000) at room temperature for 20-25 min to label the nucleus. 
	NOTE: Test the optimal dilution ratios for all the antibodies used in steps 10-11 and 13-14 in pre-experiment.</li>
	<li>Wash the retina for 3 x 30 min with 0.1% PBST on a shaker at a speed of 12- 15 rpm at room temperature.</li>
	<li>Transfer the retinal cup to a clean slide with the opening facing upward. Cut the retina radially at the 3, 6, 9, and 12 o&#39;clock positions from peripheral to central by cutting approximately 1-1.5 mm away from the optic nerve head.</li>
	<li>Add a few drops of 1x PBS to rinse the retina three times. Use air-laid paper to dry and flatten the retina. Add a drop of mounting medium (see Table of Materials) to the center of the coverslip and stop adding it until the diameter of the droplet increases to half of the coverslip. Quickly turn over the coverslip and place it on top of the outspread retina. Avoid forming bubbles.</li>
	<li>Take images of the retinal flat mounts or store and protect the slides from light at 4 &#176;C.</li>
</ol>
3. Analysis and quantification of retinal flat mounts
NOTE: For the OIR mouse model, the researchers often record the area of central retinal vascular occlusion and peripheral retinal pathological neovascularization during P12-P25. Previous studies have shown that the central avascular area of the retina reaches the maximum at P12 and gradually shrinks from P13 to P17; at the same time, the retina of OIR mice reaches the peak of neovascularization area at around P1722,29. From P17, neovessels gradually regress and functional vessels regrow into the avascular area. The retinal vasculature basically returns to normal at P2533.
<ol>
	<li>Take images of retinal flat mounts by a fluorescence microscope (see Table of Materials) with 10x objective lens. First, choose the DAPI channel and set the optic nerve head in the center of the visual field. Then, adjust other channels and focus on the superficial vasculature of the retina. Check Tiles in a photo software (see Table of Materials) and set the number of photos that need to be stitched. Click on Start Experiment to capture the whole retina.</li>
	<li>Use an image processing program (see Table of Materials) to quantify the area of vaso-obliteration (VO) and neovascularization (NV) after immunofluorescence staining.
	<ol>
		<li>First, click on the Magic Wand Tool and set an appropriate tolerance according to the difference in brightness and move the cursor to the background and click the mouse. Then, choose the Select Inverse to obtain a basic outline of the retina. Use the Lasso Tool to further outline the details of the retina. Using the Histogram function, record the pixel value of the whole retina and write it down or generate a table in a database program.</li>
		<li>Divide the retina image into four quadrants. In each quadrant, use the Lasso Tool to draw the VO area (Figure 2A-C), and use the Magic Wand Tool to select the NV area (Figure 2D-F). Through the pixel information in the histogram, calculate the pixel ratio of VO and NV to the whole retina, that is, the percentage of VO or NV area relative to the whole retina. 
		NOTE: There is also an open-source and fully automated pipeline for the quantification of VO and NV areas in OIR images using deep learning neural networks (http://oirseg.org/),&#160;which provides a reliable and time-saving way for researchers as well as unifies the standard of the quantification34.</li>
	</ol>
	</li>
	<li>Record pixel information in a spreadsheet table, which is convenient for subsequent analysis.</li>
</ol>
4. In vivo imaging with fluorescein fundus angiography (FFA)
NOTE: For OIR mice, both FITC perfusion and immunofluorescence staining can only be used for one time because of the death of experimental animals. Compared with this, one of the advantages of FFA is the observation of the dynamic changes of mouse retinal vessels during development and pathological state in vivo35,36.
<ol>
	<li>Weigh the pups before anesthesia.</li>
	<li>Anesthetize pups by intraperitoneal injection of 0.3% pentobarbital sodium at a dose of 30-50 mg/kg. 
	NOTE: For mice within 1 month, pay attention to the anesthetic doses. Use lower concentrations and doses of anesthetic to reduce the death of mice caused by anesthesia. After the pups are anesthetized, use a small heating pad to maintain the body temperature. Hypothermia not only affects pups&#39; physiological function, but also leads to changes in crystallin and accelerates the development of cataracts.</li>
	<li>Use 20 &#181;L mydriatic eye drops (0.5% tropicamide + 0.5% phenylephrine hydrochloride) for each pup and wait for 5 min to achieve long-lasting pupil dilation (Figure 3A,B).</li>
	<li>Bring the anesthetized pups in front of the imaging device (see Table of Materials). Keep the pups on a small heating pad, place the pups in a stable position, and use artificial tears regularly to maintain moisture in the cornea. Click on the mode of Infrared Fundus Imaging (IR) to adjust the optic nerve head to the center of the screen.&#160; 
	NOTE: When observing one eye of the pups, do not forget to protect the other eye. Use Hypromellose eye drops to prevent the cornea from whitening due to dryness.</li>
	<li>After intraperitoneal injection of 0.15 mL 0.5% fluorescein sodium salt solution, click on the FA button and the Injection button immediately on the touch panel of the imaging device to start timing. Record the images after 3 min when the blood circulation of the retina enters the venous phase and observe the retina no less than 6-8 min. 
	NOTE: After intraperitoneal injection of fluorescein sodium salt solution, the skin, mucosa, and urine of the pups show obvious yellowish green. Most of the fluorescein is excreted by the pups within a day. Injecting the fluorescein sodium intraperitoneally every other day for six times does not cause significant side effects37.</li>
	<li>Move the optic nerve head to the center of the image acquisition area and take the first image of the central retina. Then, move the lens of the imaging device horizontally to the nasal side of the eye until the optic nerve head is located at the midpoint of one side of the image acquisition area and take the second image. Continue to take images of the temporal, superior and inferior retina, respectively using this method (Figure 3C). 
	NOTE: Take &#34;Five-orientation&#34; images within 12 min as the regression phase occurs. The position of the optic nerve head in the inferior image is allowed not to fall on the sideline due to the limited angle adjustment of the lens.</li>
	<li>Save the images and use an image processing program for stitching.</li>
</ol>
5. Image processing of the fluorescein fundus angiography (FFA)
<ol>
	<li>Open the imaging processing program and click on New in File to create a new canvas with a black background (Figure 4A).</li>
	<li>Open an image of the central retina first in the background layer. Click on File and add the second image. Adjust the opacity of the second image to 60%, move and resize the second image until the same parts of the two images highly overlap. Click on the Switch Between Free Transform and Warp Modes button and make subtle adjustments to the vessels if necessary. Then, turn the opacity of the second image back to 100% (Figure 4A,B).</li>
	<li>Select two images at the same time and click on Auto-Blend Layers. Check Panorama as the blend method as well as select the following two sentences. Click on OK and finish the image stitching of the first two images (Figure 4C,D).</li>
	<li>Take the first two stitched images as a whole, add the third image, and continue to blend. Repeat the methods above to complete the stitching of five images (Figure 4E).</li>
	<li>Use the Crop Tool to cut images of FFA at different time points to a uniform size and observe dynamic changes of retinal vasculature from P15 to P25 in both normal and OIR pups.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Present values as mean &#177; standard deviation (s.d.).</li>
	<li>Use the Student&#39;s t-test to compare two independent samples. Use One-Way ANOVA to compare multiple sets of data and combine with Dunnett or Tukey&#39;s test, which is a commonly used multiple comparison test.</li>
	<li>For non-normally distributed data, use Mann-Whitney U test or Kruskal Wallis test. Consider significant statistical differences when P &#60; 0.05.</li>
</ol>

The authors have nothing to disclose.

The susceptibility of mice to OIR is affected by many factors. The pups of different genetic background and strains cannot be compared. In BALB/c albino mice, vessels regrow into the VO area rapidly with significant reduced neovascular tufts38, which bring some difficulties to the research. In C57BL/6 mice, there is increased photoreceptor damage when compared to BALB/cJ mouse strain39,40. The same goes for different types of transgenic mi...

Retinal neovascularization (RNV), which is defined as a state where new pathologic vessels originate from existing retinal veins, usually extends along the inner surface of the retina and grows into the vitreous (or subretinal space under some conditions)1. It is a hallmark and common feature of many ischemic retinopathies, including retinopathy of prematurity (ROP), retinal vein occlusion (RVO), and proliferative diabetic retinopathy (PDR)2.
Numerous clinical and experimental observations have indicated that ischemia is the main cause of retinal neovascularization3,4. In ROP, neonates are exposed to high-level oxygen in closed incubators to increase the survival rates, which is also an important driver for the arrest of vascular growth. After the treatment is done, the retinas of newborns experience a relatively hypoxic period5. Other situations are seen in the occlusion of central or branch retinal veins in RVO and damage of retinal capillaries is also observed which is caused by microangiopathy in PDR2. Hypoxia further increases the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) through the hypoxia-induced factor-1&#945; (HIF-1&#945;) signaling pathway which in turn guide vascular endothelial cells to grow into the hypoxic area and form new vessels6,7.
ROP is a kind of vascular proliferative retinopathy in preterm infants and a leading cause of childhood blindness8,9, which is characterized by retinal hypoxia, retinal neovascularization and fibrous hyperplasia10,11,12. In the 1950s, researchers found that high concentration of oxygen can significantly improve the respiratory symptoms of premature infants13,14. As a result, oxygen therapy was increasingly used in premature infants at that time15. However, concurrent with the widespread use of oxygen therapy in preterm infants, the incidence of ROP increased year by year. Since then, researchers have linked oxygen to ROP, exploring various animal models to understand the pathogenesis of ROP and RNV16.
In human, most retinal vasculature development is completed before birth while in rodents the retinal vasculature develops after birth, providing an accessible model system to study angiogenesis in the retinal vasculature2. With the continuous progress of the research, oxygen-induced retinopathy (OIR) models have become major models for mimicking pathological angiogenesis resulting from ischemia. There are no specific animal species in the study of the OIR model and the model has been developed in various animal species, including kitten17, rat18, mouse19, beagle puppy20, and zebrafish21. All of the models share the same mechanism by which they are exposed to hyperoxia during early retinal development and then returned to the normoxic environment. Smith et al. observed that exposing mouse pups to hyperoxia from P7 for 5 days induced an extreme form of vessel regression in the central retina and bringing them back to the room air at P12 gradually triggered neovascular tufts, which grew toward the vitreous body19. This was a standardized OIR mouse model also named as Smith model. Connor et al. further optimized the protocol and provided a universally applicable method to quantify the area of VO (vaso-obliteration) and NV (neovascularization) in 2009, which increased the acceptance and utilization of the model22. OIR mouse model is still the most widely used model now because of its small size, fast reproduction, clear genetic background, good repeatability, and high success rate.
In mice, retinal vascularization starts after birth with the ingrowth of vessels from the optic nerve head into the inner retina toward the ora serrata. During normal retinal development, the first retinal vessels sprout from the optic nerve head around birth, forming an expanding network (the primary plexus) that reaches the periphery around postnatal day 7(P7)23. Then the vessels start to grow into the retina to form a deep layer, penetrate the retina, and establish a laminar network around the inner nuclear layer (INL) as in human24. By the end of the third postnatal week (P21), deeper plexus development is almost completed. For the OIR mouse model, vascular occlusion always appears in the central retina because of the rapid degeneration of a large number of immature vascular networks in the central region during hyperoxia exposure. So, the growth of pathological neovascularization also occurs in the mid-peripheral retina, which is the boundary of the non-perfusion area and the vascular area. However, human retinal vessels have almost formed before birth. As for premature infants, the peripheral retina is not completely vascularized when exposed to hyperoxia25,26. So vascular occlusion and neovascularization mainly appear in the peripheral retina27,28. Despite these differences, the mouse OIR model closely recapitulates the pathologic events that occur during ischemia-induced neovascularization.
The induction of the OIR model can be divided into two phases29: in phase 1 (hyperoxia phase), retinal vascular development is arrested or retarded with occlusion and regression of blood vessels as a result of the decline in VEGF and the apoptosis of endothelial cells24,30; in phase 2 (hypoxia phase), the retinal oxygen supply will become insufficient under room air conditions29, which is essential for neural development and homeostasis19,31. This ischemic situation usually results in unregulated, abnormal neovascularization.
Currently, the commonly used modeling method is alternating high/low oxygen exposure: Mothers and their pups are exposed to 75% oxygen for 5 days at P7 followed by 5 days in room air till P17 demonstrated comparable results22, which is the endpoint of OIR mouse model induction. (Figure 1). In addition to simulating ROP, this ischemia-mediated pathological neovascularization can also be used to study other ischemic retinal diseases. The main measurements of this model include quantifying the area of VO and NV, which are analyzed from retinal flat mounts by immunofluorescence staining or FITC-dextran perfusion. Each mouse can be studied only once because of the lethal operation. At present, there are few methods to observe dynamic changes of retinal vasculature continuously during the process of vascular regression and pathologic angiogenesis32. In this paper, we provide a detailed protocol of OIR model induction, analysis of retinal flat mounts as well as a workflow of fluorescein fundus angiography (FFA) on mice which would be helpful to gain a more comprehensive understanding of vascular dynamic changes during two phases of the OIR mouse model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL sterile syringe</td>
			<td>Solarbio</td>
			<td>YA0550</td>
			<td>For preparation of retinal flat mounts and intraperitoneal injection</td>
		</tr>
		<tr>
			<td>1&#215; Phosphate buffered saline (PBS)</td>
			<td>Transgen Biotech</td>
			<td>&#160;FG701-01</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>2 ml Microcentrifuge Tube</td>
			<td>Corning</td>
			<td>MCT-200-C</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>48 Well Clear TC-Treated Multiple Well Plates</td>
			<td>Corning</td>
			<td>3548</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Adhesive microscope slides</td>
			<td>Various</td>
			<td></td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Adobe Photoshop CC 2019</td>
			<td>Adobe Inc.</td>
			<td></td>
			<td>For image analysis</td>
		</tr>
		<tr>
			<td>Carbon dioxide gas</td>
			<td>Various</td>
			<td></td>
			<td>For sacrifice</td>
		</tr>
		<tr>
			<td>Cover slide</td>
			<td>Various</td>
			<td></td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>World Precision Instruments</td>
			<td>14127</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>DAPI staining solution</td>
			<td>Abcam</td>
			<td>ab228549</td>
			<td>For labeling nucleus on retinal flat mounts</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olmpus</td>
			<td>SZ61</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Fluorescein sodium</td>
			<td>Sigma-Aldrich</td>
			<td>F6377</td>
			<td>For in vivo imaging</td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>&#160;Zeiss</td>
			<td>AxioImager.Z2</td>
			<td>For acquisition of fluorescence images of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Fluoromount-G Mounting media</td>
			<td>SouthernBiotech</td>
			<td>&#160;0100-01</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Hydroxypropyl Methylcellulose</td>
			<td>Maya</td>
			<td>89161</td>
			<td>For in vivo imaging</td>
		</tr>
		<tr>
			<td>Isolectin B4 594 antibody</td>
			<td>Invitrogen</td>
			<td>I21413</td>
			<td>For labeling retinal vasculature on retinal flat mounts</td>
		</tr>
		<tr>
			<td>Mice C57/BL6J</td>
			<td>GemPharmatech of Jiangsu Province</td>
			<td></td>
			<td>For OIR model induction</td>
		</tr>
		<tr>
			<td>Micro dissecting scissors-straight blade</td>
			<td>World Precision Instruments</td>
			<td>503242</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>No.4 straight forceps</td>
			<td>World Precision Instruments</td>
			<td>&#160;501978-6</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Abcam</td>
			<td>ab7475</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>O2 sensor</td>
			<td>Various</td>
			<td></td>
			<td>For monitoring the level of O2</td>
		</tr>
		<tr>
			<td>OxyCycler</td>
			<td>Biospherix</td>
			<td>A84XOV</td>
			<td>For OIR model induction</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148-1KG</td>
			<td>For tissue fixation</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Various</td>
			<td></td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Soda lime</td>
			<td>Various</td>
			<td></td>
			<td>For absorbing excess CO2 in the oxygen chamber</td>
		</tr>
		<tr>
			<td>SPECTRALIS HRA+OCT</td>
			<td>Heidelberg</td>
			<td>HC00500002</td>
			<td>For in vivo imaging</td>
		</tr>
		<tr>
			<td>SPSS Statistics 22.0</td>
			<td>IBM</td>
			<td></td>
			<td>For statistical analysis</td>
		</tr>
		<tr>
			<td>Tansference decloring shaker</td>
			<td>Kylin-Bell</td>
			<td>ZD-2008</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Tissue culture dish (Low attachment)</td>
			<td>Corning</td>
			<td>3261-20EA</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Various</td>
			<td></td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;SLBW6818</td>
			<td>For preparation of retinal flat mounts</td>
		</tr>
		<tr>
			<td>Tropicamide</td>
			<td>Various</td>
			<td></td>
			<td>For in vivo imaging</td>
		</tr>
		<tr>
			<td>ZEN Imaging Software</td>
			<td>ZEISS</td>
			<td></td>
			<td>For image acquisition and export</td>
		</tr>
	</tbody>
</table>

monitoring dynamic growth of retinal vessels in oxygen-induced retinopathy mouse model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

In the OIR mouse model, the most important and basic result is the quantification of the VO and NV area. After living in the hyperoxia environment for 5 days from P7, the central retina of the pups showed the largest non-perfusion area. Under the stimulation of hypoxia in another 5 days, retinal neovascularization was gradually produced which fluoresced more intensely than surrounding normal vessels. After P17, the fluorescence signal of pathological neovascularization regressed rapidly as the remodeling of the retina (<...

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Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...

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Research

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Biology

3.2K Views.  Sun Yat-Sen University. This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Video: Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

En Face Preparation of Mouse Blood Vessels

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the three-dimensional tissue morphology is from such sections. In addition, the sections of tissues examined may not contain the region within the tissue that is necessary for the purpose of the ongoing study. This latter limitation hinders histopathological studies of blood vessels since vascular lesions develop in a focalized manner. This requires a method that enables us to survey a wide area of the blood vessel wall, from its surface to deeper regions. A whole mount en face preparation of blood vessels fulfills this requirement. In this article, we will demonstrate how to make en face preparations of the mouse aorta and carotid artery and to immunofluorescently stain them for confocal microscopy and other types of fluorescence-based imaging.

En Face Preparation of Mouse Blood Vessels

A procedure for making en face preparations of the mouse carotid artery and aorta is described. Such preparations, when immunofluorescently stained with specific antibodies, enable us to study localization of proteins and identification of cell types within the entire vascular wall by confocal microscopy.

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...