Paulπs Immuno Protocol

Reagents etc:

Triton X 100 detergent

Phosphate buffered saline

Methanol

Goat serum

Bovine Serum Albumin

Primary antibody (Rabbit anti KI-67) Unconjugated; Polyclonal; Wide range of specificity (human, mouse, rat, pig).

Secondary, anti-Rabbit antibody (e.g. Donkey anti-Rabbit)

Diaminobenzidine

Hydrogen peroxide

Day One

1. Warm tissue for 30 min to room temperature (if previously stored at 4∞C) .

2. 3 x 10 minutes washes in 0.1 M phosphate buffer (PB) under gentle shaking at room temperature.

3. Endogenous peroxidase inhibition (if tissue has not been perfused, or not perfused well, but I do this step for all specimens as it makes the background better)

30 minutes at room temperature under gentle shaking in the following solution

Methanol 50 %

PB ≥ 50%

1.66% H₂O₂ (= (PB + methanol/30.12 = x ml of 30% H₂O₂).

4. 3 x 10 minutes washes in 0.1 M phosphate buffer (PB) under gentle shaking at room temperature.

5. Preincubation - Blocking buffer

2 hours at room temperature under gentle shaking in the following solution

0.25% Triton X in PB (stock solution)

3% Normal serum (for antibodies raised in rabbit use normal goat serum, for those raised in goat, use normal rabbit serum, etc.)

2% bovine serum albumin (BSA)

To mix the Triton X for this step, heat PB to 60∞C and add the Triton under gentle stirring. Keep stirring until clear (may take around 15 minutes). Must be at room temperature before use. Can store this in the fridge for up to one month, so I usually mix it the day before when I know how much I need (calculate enough for this step and the following step, then double it to have plenty spare in case of problems with dilutions, spills etc.).

6. Incubation in Primary Antibody

24 - 48 hours at 4∞C under gentle shaking

Same solution as above (Blocking buffer), plus appropriate dilution of primary antibody

Day Two

7. 3 x 10 minutes washes in 0.1 M phosphate buffer (PB) under gentle shaking at room temperature.

8. Secondary Antibody

1 - 2 hours at room temperature at gentle shaking in following solution

PB

3% normal serum (as appropriate)

2% BSA

Secondary antibody, appropriate dilution

I use BA 1000 or others from Vector labs as the secondary and have found a dilution of 1:500 to 1:750 gives the best result.

9. 3 x 10 minutes washes in 0.1 M phosphate buffer (PB) under gentle shaking at room temperature. Prepare AB (30 minutes before use).

10. AB incubation

Mix reactives, shake gently, and leave at least 10 minutes before use to allow the AB complex to be formed.

1 hour under gentle shaking at room temperature in the following solution

PB

A reactive, 40 µl / 5000 µl

B reactive, 40 µl / 5000 µl.

11. 3 x 10 minutes washes in 0.1 M phosphate buffer (PB) under gentle shaking at room temperature. Start to prepare DBS (20 minutes before use)

12) DAB soak (diaminobenzidine)

      5 minutes at room temperature in the following solution

      PB

      0.05% DAB

13. Reaction

To the same solution that the sections are in add 3.3 µl / ml of 30% H₂O₂.

Visually check sections for reaction. I always use a stereomicroscope to ensure that I can see neurons (that would be nuclei in the case of KI-67) before stopping the reaction.

14. 3 x 10 minutes washes in 0.1 M phosphate buffer (PB) under gentle shaking at room temperature

When I do the DAB step I usually allow 1 ml of DAB solution per section. Then I take 700 µl of this solution and soak the section in it for 5 minutes. I then add the appropriate amount of H₂O₂ to the remaining DAB solution and add 300 µl of this DAB/H₂O₂ solution to the well in which the section is