HI everyone,

I am having trouble with my immunofluorescence staining on paraffin tissue,

1) I have non-specific background staining along wiht my positive staining- i have tried to reduce primary antibody concentrations with no avail

2) my negative controls also stain albeit not as bright..

does anyone have any suggestions as to what I could try to do to eliminate these problems?

Thanks

Could you please give us the procedure that you are using for the staning

HI,

here is mr protocol:

1)Dewax and dehydrate - 3x 10 mins xylene
1x 5 mins in 100%-70% ETOH

2) Antigen retrival - Sodium Citrate 10mM 4 x 5 mins at high power in the microwave

3) Cool slides and wash 4 x 5 mins distilled water
1 x 2 mins in PBS

4) Blocking: 10% NDS in 0.1 % Tween-20 in 1 x PBS - 1 hour

5) M-20 Glucocorticoid antibody (rabbit) at 1/1000 dilution in same serum as above - o/n at RT

6)Wash - 3 x 10 mins in PBS 0.1% Tween-20

7) Secondary antibody: Donkey anti rabbit IgG conjugated to Cy3 - 1 hour

8) 3 x 10 mins washes in PBS 0.1% Tween 20

9) DAPI in mounting media- 20 mins

10) 2 x 5 mins washes in PBS

11) Mount with DACKO fluorescent m