Immunohistochemistry-Paraffin protocol specific for Collagen III Antibody (NB100-92162)

Fixing Tissue & Making Blocks:

1. After tissue has been removed, fix in 4% paraformaldehyde for two hours at 4C.
2. Transfer tissue to PBS overnight at 4C.
3. Transfer tissue to 30% sucrose for 2 hours at 4C
4. Place tissue in block as desired, fill block with OCT/embedding medium & place on dry ice until frozen.

4% Paraformaldehyde

- For 100 mL: 90mL MQ H20 and heat to 60-80C
- Measure 4 grams paraformaldehyde reagent and add to water (after reaching desired temp)
- Add 1-2 drops of 10N NaOH-to clear
- Once clear transfer to ice to cool
- After cooled, add 10mL 10X PBS
- Store at 4C
Staining with Primary Antibody:

- Wash sections in dH2O three times for 5 minutes each.
- Wash section in wash buffer for 5 minutes.
- Block each section with 100-400 ul blocking solution for 1 hour at room temperature.
- Remove blocking solution and add 100-400 ul primary antibody diluted in recommended antibody
- Diluent to each section. Incubate overnight at 4C.
- Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
- Add 100-400 ul biotinylated secondary antibody, diluted in TBST per manufacturer?s recommendation, to each section. Incubate 30 minutes at room temperature.

**If using ABC avidin/biotin method, prepare ABC reagent according to the manufacturer?s instructions and incubate solution for 30 minutes at room temperature.

- Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
- Add 100-400 ul ABC reagent to each section and incubate for 30 minutes at room temperature.
- Remove ABC reagent and wash sections three times in wash buffer for 5 minutes each.
- Add 100-400 ul DAB or suitable substrate to each section and monitor staining closely.
- As soon as the sections develop, immerse slides in dH2O.
- If desired, counterstain sections in hematoxylin per manufacturer?s instructions.
- Wash sections in dH2O two times for 5 minutes each.

Dehydrate sections:

- Incubate sections in 95% ethanol two times for 10 seconds each.
- Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
- Repeat in xylene, incubating sections two times for 10 seconds each.
- Mount coverslips.

Secondary Antibody Staining:

- Wash slides with PBT 6 x 5 minutes each.
- Add 500 uL of diluted secondary antibody, in blocking buffer**, to each slide
- Cover and incubate at room temp for 1 hour and 30 minutes
- Wash slides with PBT 6 x 5 minutes each.
- Mount slides w/ Vectashield
**Blocking Buffer (for 40 ml):
- 40 ml PBT
- 400ul HINGS (heat inactivated goat serum)
- 400ul HINDS (heat inactivated donkey serum)
- 400ul 10% Triton X-100

(When using a goat antibody, do not add HINGS)