Dissection and whole mount staining of retinas were performed as previously described [28 (link)]. Enucleated eyes were fixed for 1 h in 4% paraformaldehyde, rinsed three times in PBS, dissected and stored in methanol at −20°C. Immunohistochemistry of whole-mount samples was performed by using isolectin IB4 (Iso B4; Thermo Fisher Scientific, catalog #I21411, 1:200), rabbit anti-desmin antibody (Abcam, catalog #ab8592, 1:100), mouse anti- α-SMA (Sigma, catalog #F3777, 1:100), rabbit anti-collagen IV (Abcam, catalog #ab6586, 1:100) and rabbit anti-CD31 (Abcam, catalog # ab28364, 1:100). For detection, suitable specific Alexa Fluor-coupled secondary antibodies were used (Thermo Fisher Scientific, 1:1000). For the in vivo BrdU incorporation assay, 100 μg of BrdU (BD Pharmingen) per gram of body weight was injected intraperitoneally 4 h before the mice were euthanized with CO2 followed by cervical dislocation. Retinas were isolated and collected for analysis as above. BrdU positive cells were stained by mouse anti-BrdU antibody (Cell signaling, catalog #5292, 1:100).
Iso b4
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Iso B4 is a laboratory centrifuge designed to separate and isolate samples. It features a refrigerated system to maintain stable temperatures during the centrifugation process. The Iso B4 can accommodate a range of rotor sizes and sample volumes to meet diverse laboratory needs.
Automatically generated - may contain errors
Lab products found in correlation
Ab28364, by Abcam (2 mentions)
Mouse anti brdu antibody, by Cell Signaling Technology (2 mentions)
F3777, by Abcam (2 mentions)
Alexa fluor coupled secondary antibody, by Thermo Fisher Scientific (2 mentions)
Bromodeoxyuridine (brdu), by BD (2 mentions)
Ab8592, by Merck Group (2 mentions)
Bromodeoxyuridine (brdu), by Cell Signaling Technology (2 mentions)
Ab6586, by Abcam (2 mentions)
Alexa fluor conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Fluoro gel, by Electron Microscopy Sciences (1 mentions)
3 protocols using iso b4
1
Retinal Whole-Mount Immunostaining and BrdU Assay
2
Retinal Whole-Mount Immunostaining and BrdU Assay
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Dissection and whole mount staining of retinas were performed as previously described [28 (link)]. Enucleated eyes were fixed for 1 h in 4% paraformaldehyde, rinsed three times in PBS, dissected and stored in methanol at −20°C. Immunohistochemistry of whole-mount samples was performed by using isolectin IB4 (Iso B4; Thermo Fisher Scientific, catalog #I21411, 1:200), rabbit anti-desmin antibody (Abcam, catalog #ab8592, 1:100), mouse anti- α-SMA (Sigma, catalog #F3777, 1:100), rabbit anti-collagen IV (Abcam, catalog #ab6586, 1:100) and rabbit anti-CD31 (Abcam, catalog # ab28364, 1:100). For detection, suitable specific Alexa Fluor-coupled secondary antibodies were used (Thermo Fisher Scientific, 1:1000). For the in vivo BrdU incorporation assay, 100 μg of BrdU (BD Pharmingen) per gram of body weight was injected intraperitoneally 4 h before the mice were euthanized with CO2 followed by cervical dislocation. Retinas were isolated and collected for analysis as above. BrdU positive cells were stained by mouse anti-BrdU antibody (Cell signaling, catalog #5292, 1:100).
3
IsoB4 Staining of Brainstem Neurons
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Staining procedures were modified from previous descriptions [11 (link)]. Briefly, brainstem sections containing the MNTB were selected and incubated in a blocking solution containing (in %) 1.0 goat serum and 0.4 Triton X-100 dissolved in 0.1 M PB for 30 min at room temperature (RT). Brainstem sections were transferred to a primary label solution containing (in %) 1.0 goat serum, 0.3 Triton X-100, and biotinylated IsoB4 (1:500; Vector Labs, Burlingame, CA, USA), incubated for 2 h at RT followed by overnight incubation at 4 °C. After IsoB4 incubation, brainstem sections were washed twice in 0.1 M PB and incubated for 2 h at RT in secondary label solution containing (in %): 0.02 Triton X-100 and Alexa Fluor-conjugated streptavidin (1:500; Thermo Fisher Scientific, Waltham, MA, USA) followed by overnight incubation at 4 °C, washed 4 times in 0.1 M PB and mounted onto glass slides using a mounting medium with DAPI as a counterstain (Fluorogel, Electron Microscopy Sciences, Hatfield, PA, USA).
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