Hi, i want to study morphological changes in astrocytes using GFAP as marker. i am trying to establish a immunofluorescence method to stain astrocytes in brain sections of rats (18 um thickness) but unfortunately i am not able to get fully stained cells. some how half stained cells were detected. but still i am not satisfied with the results. the number of astrocytes in brain are good enough to get a clear picture of astrocytes during staining. please help to improve my staining method. steps followed are given below:
1. perfusion transcardialy with 0.1M PBS followed by 2% PFA.
2. Post fixation in 2% PFA at 4 degree O/N.
3. 30% sucrose at 4 degree O/N.
4. Sectioning using cryostat in OCT (18 um).
5. stored at -20.
6. Next day, kept slide at RT for 30 min.
7. maintained pH with PBS for 10 min.
8. Permeabilisation by Incubaing the samples for 10 min with PBS containing either 0.5% Triton X-100.
9. Ag retrieval by Preheat the antigen retrieval buffer (sodium citrate buffer, pH 6) to 95°C. This can be done by heating the buffer in a coverglass staining jar which is placed in a water bath at 95°C.
10. Blocking by FBS (5%) in PBS.
11. Anti GFAP Ab (Sigma) as with dilution recommended by company O/N at 4 degree (1:80).
12. Secondary Ab FITC labeled with dilution recommended by company (1:200).
13. Washing followed by mounting with (glycerol+PBS)
please find attached image.
Thank you in advance.
Hi Mohit,
First of all, always titrate your antibodies.
Is it possible for you to use an anti-fade mounting medium like Pro-Long gold? It preserves you fluorescent signal.
Furthermore I would use another fluorescent label (like Alexa-546 or Alexa 647). These will give less backgroundsignal.
With kind regards,
Ronald
Astrocytes are widely spreaded in their shape. 3-dimensional. Try thicker sections (We count the Nervecells in the Skin, or better the dendrites, because even in 50µm sections you are not able to see a nervecell in full shape)
Good Luck
Ninon
Thank you @Ronal Van for your valuable suggestions. I also did western blotting for GFAP using same antibody and titrated before blotting and calculated best concentration. In immunohisto i used three concentrations only. The minimum recommended dilution is 1:80. i also used 1:100 and 1:150 dilutions but every time getting the same results but less background in higher dilutions. please find attached data sheet for Ab i am using. i want image as attached.
I would recommand doing IHC with thicker sections (20-30 µm)and using the free floating method. The advantage is that the sections should be reacted on nboth sides. You will get a stronger result. Evan AG is not necessary. Try to make a titration with several different dilution up to 1:500 and use a secondary antibody labeled with CY2 ,CY3, Alexa488 or Alexa 546 ass Ronald has recommanded it. IF the result is not intensiv enough try to detect the primary antibody binding with biotinylated secondary antibody and Steptavidin labeled with fluophores of your choice.
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