I am performing Immunohistochemistry using superfrost plus slides (all my sections are already mounted, because I needed them also for In Situ Hybridisation).

Arround the tissue (mostly not on the tissue) there develops an intense red staining. That would be no problem if not sometimes there is a bit of this outside staining going on the tissue (see pictures) When I perform my protocol on a blanc slide (without primary or secondary antibody), just avidin-biotin-complex and AEC substrate I will have this red colour just on the "naked" slide.

My specific staining is good and I can see what I want, but sometimes there occous a reddish gradient. (see pictures)

I am really happy for any ideas!

I do not perferm a blocking step because I have no unspecific staining on my tissue, just arround!

My protocol is as following, all washing steps while shaking:

Mares endometrial biopsies

1. Rehydration

Xylene 5 min

Xylene 5 min

Xylene 5 min

100% ethanol 10 min (>99.8% pure)

100% ethanol 10 min

95% ethanol (with MilliQ H2O) 10 min

80% ethanol (with MilliQ H2O) 10 min

Water wash with MilliQ H2O 5 min

2nd water wash with MilliQ H2O 5 min

2. Antigenretrieval

2,1 g Citrat-monohydrat, pure + 900 ml MilliQ Wasser + approx. 25 ml NaOH to pH 6,0 ad 1000 ml water

(= 10 mM citric acid buffer)

20 min cooking 95 - 98 °C in cooking water bath, let cool down at RT for half an hour

3x 3 min washing in water

3. Endogenous Peroxidase blocking

10 min 3% H2O2 in MilliQ water

3 min washing in MilliQ water

Transfer to TBS

6. primary antibody (Ki-67 monoclonal, 1:3200)

- over night incubation at 4 °C

NEXT DAY

7. Wash off primary ab by 3 x 3 min in TBS

8. Secondary biotin-conjugated antibody, Incubation for 1 hour in wet chamber

9. ABC

preincubation of ABC complex for 30 min at RT

(VECTASTAIN® Elite® ABC-HRP Kit)

- 5 ml TBS + 2 drops Avidin vortex

- 2 drops biotin vortex

10. wash of secondary antibody with 3x 3min TBS

11. incubation with preincubated ABC reagent for 30 min

12. Chromogen-reaktion

fresh made "ImmPACT AEC Diluent", vortexed

• 2 Tropfen (ca. 64 ul) ImmPACT AEC Reagent 1

• 3 Tropfen (≈ 90 μl*) ImmPACT AEC Reagent 2

• 2 Tropfen (≈ 80μl*) ImmPACT AEC Reagent 3

washing off ABC with 3x 3 min TBS

then incubation with AEC and now within the first minute the glass arround the tissue starts getting red :(

13. stopping with MilliQ water

15. Counterstain

Haematoxylin 1:1 water 2 min

Tap-water 3-5x dips

Tap-water 3-5x dips

Tap-water 3-5x dips

0,02% Ammonia Water (blueing) 2-3 dips

Tap-water 3-5x dips

16. Aquatex and coverslip