Lane 1: rat brain lysates Lane 2: rat lung lysates probed with Anti DVL1/Dishevelled Polyclonal Antibody, Unconjugated (ABIN670671) at 1:200 in 4 °C. Followed by conjugation to secondary antibody at 1:3000 90min in 37 °C. Predicted band 76kD. Observed band size: 76kD.
Images provided the Independent Validation Program (badge number 029657)Formalin-fixed and paraffin embedded human kidney labeled with Rabbit Anti-DVL1 Polyclonal Antibody (ABIN670671) at 1:250 overnight at room temperature followed by conjugation to secondary antibody.
Positive control: Human kidney tissue stained with antibody
Negative control: Human colon tissue stained with antibody
Isotype control: Human kidney tissue stained with isotype control
Secondary only control: Human kidney tissue stained with secondary antibody only
Immunohistochemistry was performed on a Ventana NEXes automated platform; instrument manufacturer specific reagents are italicized.
1. Slides were preheated in convection oven at 60°C for 30 min
2. Deparaffinization procedure: - 3 changes of Xylene, 5 min each - 3 changes of 100% Ethanol, 3 min each - 3 changes of 95% Ethanol, 3 min each - Rinsed in distilled water, 3 changes
3. Heat retrieval procedure - Slides retrieved in 10.0 mM Citrate, pH6.0 in a 1000W microwave oven (~100°C) for 15 min. - Slides were allowed to cool (in citrate) for 30 min. - Slides were washed x 3 in Distilled water
4. NEXes instrument procedure, iView DAB paraffin protocol (*abridged*): - Slide chamber warmed to 37°C
5. Slides rinsed with *reaction buffer* x3
6. *iView Inhibitor (H2O2)* applied and incubated for 4 min
7. Slides rinsed with *reaction buffer*
8. Antibody Application - Primary antibody diluted 1:250 in PBS (100 microliter applied/slide) - Ventana Isotype control applied neat - Slides Incubated overnight at room temperature (~12 hours ~25°C)
9. Slides rinsed with *reaction buffer* x3
10. *iView Biotinylated IgG* applied and incubated for 8 min
11. Slides rinsed with *reaction buffer*
14. *iView Streptavidin-Horseradish Peroxidase* applied and incubated for 8 min
15. Slides rinsed with *reaction buffer*
16. *iView DAB/H2O2* applied and incubated for 8 min
17. Slides rinsed with *reaction buffer*
18. *iView Copper* applied and incubated for 4 min
19. Slides rinsed with *reaction buffer*
20. Slides washed in Dawn Detergent/tap water
21. Counterstain Procedure - Hematoxylin (Leica 560 MX) 30 sec - Slides washed in tap water, 1 min - Decolorized (10% Acetic Acid in 70% ethanol), 1 min - Slides washed in tap water, 1 min - Bluing (Austin Clear Ammonia), 1 min - Slides washed in tap water, 1 min
22. Dehydration/coverslipping procedure: - 3 changes of 95% Ethanol, 3 min each - 3 changes of 100% Ethanol, 3 min each - 3 changes of Xylene, 5 min each - Mounted with Permount
23. Imaging: Leica SCN 400F Whole Slide Scanner with Digital Image Hub and Leica Slidepath software
Step 1: Heated tissue 60°C for 30 minutes; manufacturer heats for 45 minutes.
Step 2: No ethanol wash was performed during deparaffinization; manufacturer includes 1 wash of 80% ethanol for 3 minutes.
Step 3.1: Slides were heated for 15 minutes; manufacturer provides a range of 15-20 minutes.
Step 3.2: Slides were cooled for 30 minutes; manufacturer cools for 20 minutes.
Step 4: Italicized reagents and incubation time are fixed instrument parameters.
Step 5: Secondary species-specific serum block not used; manufacturer blocks with 5% normal goat serum for 2 hours.
Step 8.1: Antibody diluted in PBS at 1:250; manufacture did not recommend diluent or dilution.
Step 8.2.1: Primary antibody incubated at room temperature overnight; manufacturer incubates overnight 4°C with agitation.