<meta charset="utf8"></head><body>小鼠全支架视网膜中 RGC 的免疫染色和基于 AI 的计数

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	<li>Quigley, H. A., Broman, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+number+of+people+with+glaucoma+worldwide+in+2010+and+2020.">The number of people with glaucoma worldwide in 2010 and 2020.</a> Br J Ophthalmol. 90 (3), 262-267 (2006).</li><li>Gallo Afflitto, G., Swaminathan, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Racial-ethnic+disparities+in+concurrent+rates+of+peripapillary+&amp;+macular+OCT+parameters+among+a+large+glaucomatous+clinical+population.">Racial-ethnic disparities in concurrent rates of peripapillary &amp; macular OCT parameters among a large glaucomatous clinical population.</a> Eye. , (2024).</li><li>Atkinson, M. 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=A+new+measure+of+patient+satisfaction+with+ocular+hypotensive+medications:+the+Treatment+Satisfaction+Survey+for+Intraocular+Pressure+(TSS-IOP).">A new measure of patient satisfaction with ocular hypotensive medications: the Treatment Satisfaction Survey for Intraocular Pressure (TSS-IOP).</a> Health Qual Life Outcomes. 1, 67 (2003).</li><li>Guo, 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=A+new+mouse-fixation+device+for+IOP+measurement+in+awake+mice.">A new mouse-fixation device for IOP measurement in awake mice.</a> Vision Res. 219, 108397 (2024).</li><li>Almasieh, M., Wilson, A. M., Morquette, B., Cueva Vargas, J. L., Di Polo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+retinal+ganglion+cell+death+in+glaucoma.">The molecular basis of retinal ganglion cell death in glaucoma.</a> Prog Retin Eye Res. 31 (2), 152-181 (2012).</li><li>Jeon, C. J., Strettoi, E., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+cell+populations+of+the+mouse+retina.">The major cell populations of the mouse retina.</a> J Neurosci. 18 (21), 8936-8946 (1998).</li><li>Zhang, 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=Automatic+counting+of+retinal+ganglion+cells+in+the+entire+mouse+retina+based+on+improved+YOLOv5.">Automatic counting of retinal ganglion cells in the entire mouse retina based on improved YOLOv5.</a> Zool Res. 43 (5), 738-749 (2022).</li><li>Al-Khindi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+Tbx5+regulates+direction-selective+retinal+ganglion+cell+development+and+image+stabilization.">The transcription factor Tbx5 regulates direction-selective retinal ganglion cell development and image stabilization.</a> Curr Biol. 32 (19), 4286-4298 (2022).</li><li>Claes, M., Moons, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+ganglion+cells:+Global+number,+density+and+vulnerability+to+glaucomatous+injury+in+common+laboratory+mice.">Retinal ganglion cells: Global number, density and vulnerability to glaucomatous injury in common laboratory mice.</a> Cells. 11 (17), 2689 (2022).</li><li>Zhang, N., Wang, Z., Lin, P., Xing, Y., Yang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methanol-based+whole-mount+preparation+for+the+investigation+of+retinal+ganglion+cells.">Methanol-based whole-mount preparation for the investigation of retinal ganglion cells.</a> J Vis Exp. (194), e65222 (2023).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1&#215; PBS</td>
			<td>Servicebio</td>
			<td>G4202</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa594-conjugated Goat Anti-Rabbit IgG&#160; (H+L)</td>
			<td>ThermoFisher</td>
			<td>A-11012</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-BRN3A antibody [EPR23257-285]</td>
			<td>Abcam</td>
			<td>ab245230</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoCount software</td>
			<td></td>
			<td></td>
			<td>https://github.com/MOEMIL/Intelligent-quantifying-RGCs</td>
		</tr>
		<tr>
			<td>Cloud disk</td>
			<td>Google drive link</td>
			<td></td>
			<td>https://drive.google.com/file/d/1yOEsBvil6KEdZFa5ENQxB6&#160;</td>
		</tr>
		<tr>
			<td>Cloud disk</td>
			<td>Baidu link</td>
			<td>Extraction code: g44k</td>
			<td>https://pan.baidu.com/s/1lccg1OVbeudsp2VtnqxWZg&#160;</td>
		</tr>
		<tr>
			<td>Marker</td>
			<td>Sharpie</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Biosharp, Labgic</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Macklin</td>
			<td>P804536</td>
			<td></td>
		</tr>
		<tr>
			<td>ProClean 300</td>
			<td>Beyotime</td>
			<td>ST853</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>BBI, Sangon</td>
			<td>A610498</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BioFroxx, neoFroxx</td>
			<td>1139ML100</td>
			<td></td>
		</tr>
	</tbody>
</table>

whole-mount immunostaining and automatic counting of mouse retinal ganglion cells

Glaucoma is characterized by&#160;the progressive death of ganglion cells, which&#160;poses a significant challenge for sight&#160;restoration1,2. This disease is a major focus of ophthalmic research due to its prevalence and impact on vision3. Mouse models are indispensable in glaucoma 
research due to&#160;their homogeneous genetic background, high&#160;reproductive capacity, and the similarity of&#160;their ganglion cell properties to humans4. The primary goal of this method is to accurately quantify retinal ganglion cells (RGCs) in mouse models, which is essential for understanding the pathogenesis of glaucoma and developing relevant treatments.
The rationale behind developing this technique stems from the need for a reliable and efficient method to assess RGC degeneration in mouse models. Traditional methods, such as labeling RGCs in retinal sections, often provide unreliable results due to the non-uniform distribution of RGCs in the retina5. Quantifying RGCs across the entire retina better reflects changes in their numbers and is crucial for evaluating disease progression and therapeutic interventions.
This method offers several advantages over alternative techniques. For instance, manual counting of retinal ganglion cells (RGCs) in a normal adult mouse retina, which contains 40,000 to 60,000 RGCs, can be time-consuming and error-prone6,7,8. The software for automatic RGC counting that we developed allows for accurate counting in less than 3 min, potentially saving researchers a significant amount of time. Additionally, the AI-based software used&#160;for automatic counting minimizes bias and&#160;enhances reproducibility.
Furthermore, this technique provides a standardized approach to assess RGC degeneration across different mouse models and experimental conditions, contributing valuable data to the field of glaucoma research. The method aligns with other studies that emphasize the importance of whole-mount retina analysis for understanding retinal changes in diseases9.
To help readers determine whether this method is suitable for their application, it is important to note that this technique is particularly advantageous for researchers studying retinal ganglion cell (RGC) degeneration in mouse models of glaucoma or other retinal diseases. The method is adaptable to various experimental setups and offers a high degree of accuracy and efficiency in RGC counting, making it ideal for both small-scale and large-scale studies. Additionally, the protocol&#39;s straightforward design and the availability of user-friendly software make it accessible to researchers with varying levels of expertise in retinal analysis.

<meta charset="utf8"></head><body>作者聚焦：青光眼小鼠模型中的高效视网膜神经节细胞计数用于治疗评估

小鼠视网膜神经节细胞的全支架免疫染色和自动计数

Our research scope is to use the mouse as an animal model for studies of glaucoma. To precise assess the disease condition of glaucoma in the mouse retina, we need to determine how many retinal ganglion cells are in the retina. There around the 50, 000 retinal ganglion cells in an entire mouse retina.If we count them manually, it may take up to eight to 10 hours. Automatic counting methods can get it done in minutes and save people lots of time and effort. In the future, our lab will focus on glaucoma treatment study.We are going to use different mouse models for glaucoma to test a variety of treatment methods. The method described in this protocol is particularly useful for treatment evaluation.

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve ...

whole-mount-immunostaining-automatic-counting-mouse-retinal-ganglion

Research

JoVE Journal

Neuroscience

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

442 次观看.  University of Electronic Science and Technology of China. 我们的研究范围是将小鼠作为青光眼研究的动物模型。为了精确评估小鼠视网膜中青光眼的疾病状况，我们需要确定视网膜中有多少视网膜神经节细胞。整个小鼠视网膜中大约有 50， 000 个视网膜神经节细胞。如果我们手动计数，可能需要长达 8 到 10 小时。自动计数方法可以在几分钟内完成，并为人们节省大量时间和精力。未来，我们的实验室...