It would help if you can attach an image of your staining.

As you use Goat and Donkey, each 0.5% for both the blocking solution and diluting your antibody, I suggest using a higher percentage of goat/donkey serum for your blocking solution and lower one for the one you are going to dilute your antibody in. The same goes for the secondary antibody.

Concerning your washing, you can always increase the number of times you wash the slides. Mounting medium, I'm not sure if it matters.

I agree with Vahid M. Harandi that 2.5 or even 5% BSA, Goat, or Donkey serum is preferable. Also since sections are fairly thick I would suggest blocking overnight at 4C. You also missing the permeabilization step before blocking for your section thickness it would be about 30 minutes in 0.5% Triton X it might not be required if you are looking for the cell surface proteins. I also think your time in citrate buffer is too long 15-20 minutes is usually sufficient. Keep in mind that brains are fairly autofluorescent at 488 nm so your secondary should avoid this wavelength the best is 647nm/CY5. I believe by increasing blocking solution concentration and using 647/Cy5 secondary you will get publishable quality data.

Hello,

Please follow this procedure for optimal staining of the brain samples:

1. Section the slides at 5-10µM thickness. *** 50µM is too thick and it will cover the overall details of the brain slide.

Deparaffinization:

2. Xylene-Xylene-100%EtOH-100%EtOH-96%EtOH-75%EtOH each for 5 min

3. Wash under running tap water for 5 minutes

4. Dip the slides in antigen retrieval buffer and heat slide in a coupling jar for high voltage (700W) for 10 min (Start with low voltage to heat it and increase it to high voltage and continue for 10 min: *** open the MW door when you see the liquid is boiling>avoid overflow.

5. Keep the C.jar under running tap water for 10 minutes.

6. Permeabilize cells in 1% Triton X-100 for 15min in RT

7. Rinse Slide X2 in 1X PBS:5 min each washing step

8. Incubate Slide in 3% methanolic H2O2 for 30min (blocking of endogenous peroxidase activity)

*** This step will greatly reduce the final background staining of your samples

9. Rinse Slide X2 in 1X PBS :5 min each washing step

10. Blocking: Incubate with 5% goat serum at 1-hour RT (5% goat serum can be made in 1X PBS solution)

11. Drain off the blocking buffer

12. Add primary antibody (optimize the dilution: start with 1:100; in 5% goat serum) and incubate it in humidified chamber overnight at 4C

*** Humidified chamber can be made from empty slide box bottom filled with wetted tissue papers

13. Drain off antibody and Rinse Slide X2 in 1X PBS :5 min each washing step

14. Incubate with secondary antibody for 1hour

15. Drain off antibody and Rinse Slide X2 in 1X PBS :5 min each washing step

16. Incubate with Streptavidin-HRP conjugate (1:250 in PBS) at RT for 1-hour *** If you need clear and clean staining, add this step.

17. Wash with TBSX2; 5 min each

18. Incubate with H2O2-DAB solution under a microscope; check for optimal

19. Slides should keep in Distilled water after DAB development

20. Wash under running tap water for 5min> Wash with distilled water X3; 5 min each> Counterstain in Hematoxylene; dip once in Hematoxylene> Wash under running tap water for 5 min>

21. Dehydrate the tissue sections with four changes of ethanol (95%/95%/100% and 100%);3 min each> Clear the slides in xylene (xylene 1 and 2)> Mount slides with DPX mount medium.

Hope this helps

Charith

Thank you Charith UB Wijerathne Anton Lennikov and Vahid M. Harandi for replying. Your suggestions really helped in optimizing my experiment.

Kind regards,

Farwa