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.