Immunohistochemistry protocol

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Immunohistochemistry protocol

Fig. The schematic diagram of immunohistochemistry.

Here we provide a protocol for usual immunohistochemistry.


  1. Tissue samples are dissected from animals follow an appropriate protocol.
  2. Samples are immediately fixed in Carnoy’s solution and left overnight.


  1. The following day, the tissues are embedded into paraffin. To do so, each sample is washed with ethanol 4X for 45 minutes each time to remove the chloroform.
  2. Each sample is cleaned with xylene two times for 30 minutes each time at 58°C.
  3. The samples are infiltrated with paraffin 3 times for 20 minutes each at 58°C.
  4. The samples are then stored at room temperature overnight so that they would be ready the next day for sectioning.


For immunofluorescence labeling of organ tissue, a paraformaldehyde fixation and paraffin embedding protocol is used. The organs are obtains by dissection.

  1. After the tissues are embedded in paraffin, a few blocks are placed in the freezer so they will be easier to section, and to avoid breaking the blocks.
  2. After 15 minutes in a freezer, the blocks are sectioned into 5 µm sections with the microtome and mounted onto slides.
  3. These are left overnight to dry.


  1. Slides are placed into the oven for 30 minutes.
  2. Slides are placed in xylene for 2 minutes (X2) in order to dissolve the paraffin.
  3. The rehydration process is then begun using 100% ethanol for 1 minute, then 95% ethanol for 1 minute, than 70% ethanol for 1 minute, and finally 50% ethanol for 1 minute.

Enzyme reactions

  1. Then, slides are washed with PBS/Tween 20 for 5 minutes and the slides are then dried and marked with the pap pen in order to keep the future solutions on the slide.
  2. 200 µL of PBS/proteinase kinase (PBS-PK) solution is applied onto each slide.
  3. The slides are incubated in a closed box for 15 minutes, and are then washed with PBS/Tween 20 for 5 minutes.
  4. Slides are then placed in peroxidase solution for 10 minutes (90% methanol/20% hydrogen peroxide) to open up the cell membranes.

Primary antibody binding

  1. The slides are once again washed with PBS/Tween 20 for 5 minutes (3 times).
  2. They are then placed inside a box to which water and paper are added to maintain moisture.
  3. A Zymed IHC kit is used and reactions are performed following manufacturer’s specifications as follows. A serum blocking solution is applied for 10 minutes. The primary antibody is prepared at dilutions of 1:100 and 1:2,000 respectively, and 200 µL of each primary antibody applies to the respective slides.
  4. These are incubated at 4°C freezer overnight.

Secondary antibody binding and coloration

  1. The next day, the slides are rinsed using PBS/Tween 20 for 2 minutes (3 X).
  2. Two drops of secondary antibody is then applied to each slide.
  3. After incubating for 10 minutes, the slides are rinsed with PBS/Tween 20 2 minutes (3 X).
  4. The AEC chromogen is then used follow the manufacturer’s protocols.
  5. After incubation, the slides are rinsed with distilled water, mounted, and pictures are taken.

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