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Mouse Anti B-gal in paraffin embedded sections

Streptavidin FITC or TexasRed

1. Deparaffinize 20minX2. Rehydrate slides to ddH₂O
2. Wash in ddH₂O 3min X2.
3. Unmasking:
   Set water bath to 95-99°C and prewarm 200ml ddH₂O.
   Add 1.9 ml Antigen Unmasking Solution (Vector) to 200ml ddH₂O.
   Leave in water bath for 20 min at 95-99°C.
4. Wash in ddH₂O 1min X2.
5. Wash in 0.1% PBST ( PBS + 0.1% Tween 20) 3min X2.
6. Block non-specific proteins with 10% goat serum in 0.5% PBST
   ( PBS + 0.5% Tween 20) for 30-60 min at RT.
7. Incubate with Mouse anti B-gal Ab (Promega Z378A)1:50 in 5% goat serum in PBST O/N at 4°C.
8. Wash slides with 0.1% PBST 5min X3.
9. Incubate with 1:200 Biotinylated goat anti-mouse IgG (Vector) in 0.1% PBST for 1hour at RT.
10. Wash slides with 0.1% PBST 5min X3.
11. Incubate with 1:200 Streptavidin FITC (NEN) ) in 0.1% PBST for 1hour at RT.
12. Wash slides in PBS for 5minX3.
13. Cover slides with Vectashild mounting medium (Vector).

 

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