IHC Protocol Frozen sections

The general guideline for IHC frozen fixed sections has been outlined below.

Frozen Fixation

1) Place sample in tissue mold with frozen tissue matrix.

2) Cover the tissue with cryo-embedding media.

3) Lower the tissue mold into liquid nitrogen, ensuring the sample is frozen completely.

4) Store the block at -80 until required for testing.


Cutting the sample

1) Transfer the tissue block into the cryostat. Allow the sample to equilibrate to the temperature of the cryostat which is usually around 20 C.

2) Using a cryotome section the samples into 5-10 um slices.

3) Place the cut samples onto a glass slide. Allow to dry at room temperature overnight for the sample to adhere to the slide.

4) Place slide in cold acetone (-20) for 5 minutes to fix sample.

5) Remove from the acetone and allow the slide to dry for one hour.

6) Rinse slides in PBS for 5 minutes, repeat twice.


Staining the sample

1) Incubate the slide in 0.3 % H2O2 and PBS for 10 minutes.

2) Rinse the slide in PBS for 5 minutes, repeat twice.

3) Incubate the slide with blocking buffer for 30-60 minutes.

4) Remove the slide from the blocking buffer and apply the primary antibody. Incubate for 1 hour at room temperature or 4C overnight.

5) Rinse the slide in PBS for 5 minutes, repeat twice.


6) Dilute the secondary antibody with antibody buffer.

7) Apply the secondary antibody to the slide and incubate for 30 minutes.

8) Rinse the slide in PBS for 5 minutes, repeat twice.


9) Apply an appropriate conjugate for the secondary antibody.

10) Rinse the slide in PBS for 5 minutes, repeat twice.


11) Prepare the required DAB staining solution, apply to slides and incubate for 5 minutes.

12) Rinse the slide in PBS for 5 minutes, repeat twice.

At this stage a counter stain may be applied, if required. Then rinse the tissue in water for 15 minutes.


Dehydrate sample

1) 95% alcohol for 5 minutes, repeat once.

2) 100% alcohol for 5 minutes, repeat once.


Addition of cover-slip

1) Place slide in xyelene, repeat three times.

2) Use mounting solution to adhere coverslip.

Slide may then be observed under a microscope.