Quantification of ZNF536 was performed using an image analysis procedure described elsewhere extensively34 (link). Briefly, Image Proversion 6.3 (Media Cybernetics, Rockville, USA) was used. A black and white camera (SONY XC-77E) was mounted on a microscope (Zeiss Axioskop with Plan-NEOFLUAR Zeiss objectives, Carl Zeiss GmbH, Jena, Germany). The SCN (area total) was outlined by the AVP staining and captured with at a ×20 objective. The outline of the SCN was then transferred to the adjacent ZNF536 stained image. The threshold for the positive signal was set to twice the mean optical density (OD) of the background and a mask of the stained structures was made. The computer determined the OD of the mask and surface area covered by the signal (area mask). Multiplying the OD by the area mask gave the integrated optical density (IOD). Subsequently, the final parameter was the cOD, which was calculated by dividing the IOD by the representative area of the SCN. For each subject, the cOD was used to describe the average concentration of ZNF536-ir in the SCN section.
Imagepro version 6
Manufactured by Media Cybernetics
Sourced in United States
ImagePro Version 6.0 is a software product developed by Media Cybernetics. It is a comprehensive image analysis and processing solution designed for various applications. The software provides a range of tools and features for tasks such as image capture, enhancement, measurement, and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Axioskop, by Zeiss (1 mentions)
Plan neofluar objective, by Zeiss (1 mentions)
Fluorescent microscope, by Leica (1 mentions)
Stereotactic apparatus, by Stoelting (1 mentions)
Superfrost plus microscope slides, by Avantor (1 mentions)
Vectamount, by Vector Laboratories (1 mentions)
Formaldehyde, by TAAB (1 mentions)
5 protocols using imagepro version 6
1
Quantitative Analysis of ZNF536 in SCN
2
Quantitative Image Analysis of SCN Neurons
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Quantitative image analyses were carried out by one investigator (X-Y Wu), who was blind to the diagnosis and experimental conditions. Quantification of the integrated optical density (IOD) of immunocytochemistry or in situ hybridization signals was performed as described in our previous studies (Zhu et al. 2016 ). The setup consisted of an image analysis system (Image Proversion 6.3, Media Cybernetics, Rockville, USA) connected to a black and white camera (SONY XC-77E) mounted on a microscope (Zeis Axios-kop with Plan-NEOFLUAR Zeiss objectives, Carl Zeiss GmbH, Jena, Germany). The SCN area covered by AVP-stained neurons was outlined manually at a 20× objective. The outline of the SCN was then transferred to the adjacent GAD65/67-ir or GAD67-mRNA stained image by selecting at least four corresponding marker points in both images (see Fig. 2 a, c). For data collection and calculation, see Methods in Supplement.
3
In vivo Retrograde Labeling of Retinal Ganglion Cells
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To assess the effect of DHPG on RGCs in vivo, retrograde labeling of RGCs was performed. After anesthetization, the rats were placed in the stereotactic apparatus (Stoelting, Wood Dale, IL, USA) and the brain surface was exposed by perforating the parietal bone to facilitate dye injection. 2 μL of 2% fluorogold (FG; Biotium, Hayward, CA, USA) was injected into both superior colliculi and dorsal lateral geniculate nuclei. After seven days, rats from different groups were sacrificed and eyeballs were enucleated and placed in 4% paraformaldehyde for 4 h. The whole retina was then carefully dissected, flattened, and mounted with the vitreous side up on slides. Photographs were captured using a fluorescent microscope (Leica, Germany) and FG-labeled RGCs were counted in a masked fashion by the same investigator using automated particle counting software in ImagePro Version 6.0 (Media Cybernetics, Bethesda, USA). The number of labeled cells in 12 photographs of each retina (three photographs per retinal quadrant) at 1/6, 3/6, and 5/6 of the retinal radius was summed together and expressed as mean RGC densities/mm2 for each group.
4
Retinal Ganglion Cell Quantification
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Nineteen days after ONC, FG (Cambridge Biosciences, Cambridge, UK) was injected into the optic nerve, between the lamina cribrosa and the ONC site (Vigneswara et al., 2013, 2014 ). Animals were killed 2 days later and the dissected retinae were immersion-fixed in 4% formaldehyde (TAAB Laboratories, Aldermaston, UK), flattened onto Superfrost Plus microscope slides (VWR International, Lutterworth, UK) by making four equidistal radial cuts to obtain four quadrants, air dried and mounted in Vectamount (Vector Laboratories). The identity of individual retinae was anonymised by a second investigator before image capture using a Zeiss epifluorescent microscope and the number of FG+ RGCs counted in ImagePro Version 6.0 (Media Cybernetics) from captured images of 12 rectangular areas, 3 from each quadrant, placed at radial distances from the centre of the optic disc of the inner (1/6 eccentricity), midperiphery (1/2 eccentricity) and outer retina (5/6 eccentricity). The mean densities of FG+ RGCs/mm2 for each retina were determined.
5
Histomorphometric Analysis of Renal Fibrosis
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Left kidneys were fixed in formalin and embedded in paraffin, sectioned at 5 µm and processed using our published methods (Sankaran et al. 2007) . For quantification of tubulointerstitial fibrosis, sections were stained with Sirius red (an adaptation of Masson's trichrome stain), which permits image analysis measurement using a standard incandescent microscope light source. 10X magnification images from a standard microscope were captured with a Spot Junior chargecoupled device camera by random stage movement throughout the renal cortex and were analyzed with Image Pro version 6.0 (Media Cybernetics, Silver Spring, MD, USA). An average of twenty-six images per kidney were collected for all histomorphometric analyses, and all measurements were carried out in a blinded fashion. Tubulointerstitial fibrosis was measured by densitometry as described (Sankaran et al. 2004 ). Transverse tissue sections were stained with hematoxylin and eosin for glomerular diameter measurement and mean glomerular volume was determined with standard stereological techniques developed by Weibel, as described previously D r a f t 8 (Sankaran et al. 2007 , Caligiuri et al. 2014) . Structural damage in the glomeruli was analyzed using an objective scoring system, as previously described (Caligiuri et al. 2014) .
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