I am doing immunofluorescence staining on retinal sections from RCS rats to assess GFAP. As shown in my attached image, the GFAP staining is not consistent with most literature I've seen with retinal GFAP. Typically, I see long connected processes throughout the length of the retina following injury. But as shown in my image, I get short processes. If anyone has any information on if I am doing something wrong or explanation about the images, I would greatly appreciate help.
Here are all the details that might help:
- RCS rats are a retinal degeneration model, they were p90 when euthanized
- fixed with Davidson's (formaldehyde based), paraffin embedded, cut at around 7-10 microns (I think 7)
- did heat antigen retrieval using citrate buffer at pH 6 (I know I probably don't need to but I have to for a co-stain I will use in the future)
- blocking buffer is 5% goat serum and 0.1% triton-X in PBS, GFAP antibody is rabbit polyclonal (dako/agilent z0334) diluted 1:1000 in blocking buffer (I did a titration all look similar in terms of structure of GFAP), and secondary is goat anti-rabbit conjugated with alexa fluor 594 (invitrogen, A-11037 ) diluted 1:500 in blocking buffer
This is very normal Kabir. I see similar patterns after injury. These short processes do occur and could be due to the thickness of your sectioning. Nothing to worry about.
Andrew Osborne Just wanted to add, after talking with colleagues they mentioned the pattern is related to the section cutting angle so the glial fiber is likely cut/broken on this sections. Other slides or eyes will have differing patterns. Hope that adds to the possibilities.
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