Abcam Alpha Smooth Muscle Actin Antibody (ab7817)

This antibody can be found on the Abcam website ab7817.

This antibody was tested in 2015 with mouse colon and human hepatitis tissues.

The protocol is fairly straight forward. All of the testing is done by hand.

This is a mouse monoclonal antibody. If you are staining mouse tissues, then using a mouse blocking protein like Vectors mouse on mouse kit is very helpful to block unnecessary staining.

Antibody SMA Protocol                                                                    12/14/2015

Equipment and Reagents

  • 2 changes of xylene and 100% alcohol and 1 change of both 95% alcohol and 70% alcohol
  • Distilled water
  • Abcam Primary Antibody – alpha SMA Abcam – ab7817 Dilution 1:50
  • VECTOR MOM ImmPRESS KIT (Peroxj-dase Polymer) (MP-2400)
  • Vector DAB
  • 1X PBS
  • 1X PBST
  • Fresh 3% Hydrogen peroxide
  • Pressure cooker
  • Sequenza staining racks or multiple IHC humidity chambers
  • Disposable pipets
  • 1 – Drummond pipetor
  • Multiple micro pipetors with tips
  • Mouse positive control tissues; Intestine or testicle, ovary or artery.

Heat-mediated antigen retrieval using citrate buffer, pH 6.0

Citrate buffer (10 mM citric acid, pH 6.0)

Citric acid (anhydrous), 1.92 g
Distilled water, 1000 ml
Mix to dissolve 
Adjust pH to 6.0 with 1 M NaOH (be sure to mix well) 
Store this solution at room temperature for 3 months, or at 4°C for longer usage

Method

Start

  1. Run slides to distilled water.
  2. 5 slides – HIER using sodium citrate pH 6.0 solution in pressure cooker (steam) for 10 minutes heat up, 10 minutes to cook, 10 minutes under running water in the cooker (lid on) and 10 minutes running water (lid off).
  3. Block with fresh 3% H2O2 – 15 minutes
  4. Wash – PBST – 2 X 5 minutes
  5. Block in M.O.M Ig serum – 60 minutes
  6. Wash in PBST 2 X 2 minutes
  7. Incubate in M.O.M 2.5% normal horse serum – 10 minutes
  8. Aspirate blocking (do not wash) and incubate in primary antibody (Abcam – ab7817) 1:50 diluted in M.O.M 2.5% horse serum – 30 minutes.
  9. Wash – PBST 2 X 2 minutes.
  10. Incubate in M.O.M impress anti-mouse Ig reagent (blue label bottle) – 10 minutes.
  11. Wash PBS 2 X 5 minutes.
  12. Prepare DAB.
  13. Apply 50-200 ul to positive controls and watch under microscope – average 1 minute, 15 seconds.
  14. Wash in distilled water 5 minutes.
  15. Pass Hematoxylin solution through 0.2 mm filter
  16. Repeatedly dip slides in Hematoxylin for 30 seconds
  17. Rinse with water by dipping in water bath for 10 seconds
  18. Submerge in bath of 0.1% sodium bicarbonate solution for 30 seconds
  19. Rinse with water by dipping in water bath for 5 seconds
  20. Submerge slides in running water for 5 minutes
  21. Dehydrate in 2 changes of 95% alcohol, 100% alcohol and clear in 2 changes of xylene.
  22. Coverslip

Results: Intracellular

Download it here:

Due to discrepancies between our protocol and the Abcam website data, we suggest you use our information as reference material only.