IHC Protocol FFPE

The general guideline for IHC formalin fixed paraffin embedded sections has been outlined below.

Formalin Fixation

1)  Collect tissue samples rinsing in PBS to remove any blood.

2) Place the samples in buffered formalin ( 3.7% formaldehyde and 10 mM of phosphate buffer pH 7.4)

Incubate the samples for 24-48 hours. Please ensure plenty of fixative is used to cover the tissue, fixative volume should be 5-10 times that of the tissue volume.

3) Rinse the tissues for 1 hour in running water.


Parraffin Embedding

Place in increasing concentrations of alcohol to dehydrate the sample.

1) 70% ethanol for 1 hour, repeat twice.

2)  80% ethanol for 1 hour, repeat once.

3) 95% ethanol fror 1 hour, repeat once.

4) 100% ethanol for 1.5 hours, repeat 3 times.

Xylene and Paraffin are then used to replace the alcohol.

5) Xylene for 1.5 hours, repeat 3 times.

6) Paraffin wax for 2 hours at 60 C, repeat twice.


Cutting tissue and mounting

1) Paraffin embedded tissue may then be trimmed using a microtome, cutting into 3-10 um slices.

2) Place in water bath at 40-45 C

3) Mount sections onto slides.

4) Air dry the slides for 30 minutes

5) Bake the slides at 45-50 C overnight, do not allow the temperature to rise above 50 C as this can cause the slides to crack.

6) Place the slide in Xylene for 10 minutes repeating 2-3 times.


Rehydrate sample placing in decreasing concentrations of ethanol.

1) 100% ethanol for 3 minutes,

2) 95% ethanol for 1 minute,

3) 80% ethanol for 1 minute,

4) Rinse in distilled water.

At this stage an antigen retrieval method may be carried out if required.


Staining of tissue

This process should be carried out in a humiified chamber. Do not let the samples dry out at any point.

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


2) Dilute the primary antibody with antibody buffer .

3) Remove the slide from the blocking buffer and apply the primary antibody. Incubate for 1 hour.

4) Rinse the slide in PBS for 2 minutes, repeat 3 times.


5) Dilute the secondary antibody with antibody buffer.

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

7) Rinse the slide in PBS for 2 minutes, repeat 3 times.


8) Apply an appropriate conjugate for the secondary antibody.

9) Rinse the slide in PBS for 2 minutes, repeat 3 times.


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

11) Transfer the slides a glass dish. Add 5 ul of 0.3% H2O2. The incubation time is dependent upon the concentration of the target.

12) Under a microscope observe the colour development. PBS should be used to halt the reaction.

13) Rinse the slide in PBS for 2 minutes, repeat 3 times.

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.