Login Register


$ 0

Immunohistochemical project | solve all immunohistochemical problems here

Date:2023-06-02 Views:496

Congratulations! Click on this article, and you're halfway to your immunohistochemical experiment!

Let's move on to the subject of today's discussion: immunohistochemistry

Common Staining Methods for Immunohistochemistry

Hematoxylin-Eosin (HE) Staining

Hematoxylin-Eosin staining (HE) was one of the common staining methods for paraffin sectioning techniques. The main staining principle is that hematoxylin dyeing solution is alkaline, which can make the chromatin in the nucleus and nucleic acid in the cytoplasm appear blue. Eosin is an acidic dye that causes components in the cytoplasm and extracellular matrix to appear red. HE staining is the most basic and widely used technique in the teaching and research of histology, embryology and pathology.

Diaminobenzidine (DAB) Staining

Different from HE staining, DAB staining can detect protein expression in specific cells based on specific cell markers. The primary antibody is used to bind to the target protein antigen in the tested tissue, and then the secondary antibody labeled by HRP is used to bind to the primary antibody. Finally, the localization and semi-quantification of the protein antigen to be tested are confirmed by reacting with DAB chromogenic agent. DAB (diaminobenzidine) stain stains horseradish peroxidase brown.


Although the principle of the experiment seems simple, the reality is not always imagined……

Experiment Tips

In order to ensure the accuracy and reliability of the experimental results, please check whether your experimental design meets the following requirements before the experiment begins

  1. Negative control and positive control were set in each group
  2. Determine if the target antigen is only expressed at a specific site.

OK, now we are going to exhibit the results of our daily experiments

Common Experimental Problems

  1. All the radiographs were negative, including positive controls.
  2. All the sections showed positive results, including negative control.
  3. The section has a high background.
  4. The positive control was stained well, and the positive specimens tested showed negative results.

Reasons Analysis and Suggestions

The biopsy showed positive results

  1. The antibody concentration was too high during the staining process, or the slices dried out.

It is suggested to reduce the antibody concentration, keep the section moist, set the antibody concentration gradient for the first experiment, and select the optimal conditions.

  1. Buffer solution configuration is not added sodium chloride and pH value is not accurate, washing is not thorough.

It is recommended to check the buffer pH and wash fully.

  1. The use of discolored substrate solutions, or color reaction time is too long.

It is recommended to replace the new color substrate.

  1. The incubation time of the antibody is too long.

It is recommended to shorten the incubation time of antibody appropriately.

  1. The concentration of H2O2is too high, the color rate is too fast and the adhesive is too thick.

It is recommended to reduce the concentration of H2O2, control the color speed and clean in time.

The background of the section is too deep

  1. Endogenous peroxidase was not completely blocked.

It is recommended to use H2O2 for sample pretreatment.

  1. The section or smear is too thick.

It is recommended to adjust section or smear thickness.

  1. Rinsing is not enough.

Full rinsing is recommended, which can be rinsed for short periods of time and repeated several times.

  1. Substrate color reaction is too long.

It is suggested to observe the color effect at all times and terminate the reaction in time.

  1. Insufficient protein blocking or use of serum hemolysis.

It is suggested that serum from the same species of the second antibody should be used for sample sealing and the sealing time can be extended appropriately. For multiple immunofluorescence, secondary antibodies treated with serum preadsorption can be selected to reduce background nonspecific binding.

  1. The use of whole-serum antibody dilution is insufficient.

It is suggested to set the concentration gradient and select the best condition for the first experiment.


The staining of the slides is uneven

  1. Insufficient dewaxing.

It is recommended to bake at 60℃ for 20min and immediately put in fresh xylene.

  1. Inadequate hydration.

It is recommended that fresh gradient ethanol should be prepared frequently.

  1. The antibody is not mixed.

It is recommended to use a pipette gun to thoroughly mix primary antibody/secondary antibody and other reagents.

  1. Slant the section when the antibody is incubated.

It is recommended to keep the section level and incubate evenly.

  1. Inadequate PBS washing after antibody incubation.

It is recommended to rinse fully. Rinse for a short period of time and repeat several times.

  1. Uneven thickness of production and other problems.

It is recommended to set the section thickness uniformly.

  1. The dyeing box is uneven and the slices tilt.

It is recommended to maintain the section level and incubate evenly.

The positive control was stained well, and the positive specimens tested showed negative reaction

  1. Improper fixation and handling of specimens.

It is recommended to reprepare the sample.


After reading the above reason analysis and suggestions, do you have a clearer optimization direction for the immunohistochemical experiment? Get moving and start your new immunohistochemical experiment!


Abbkine IHC/ IF series products make your immune testing more convenient and less worrying!

Related Product Recommendation

Cat. No. Product Name Size
KTD107-EN Universal IF Toolkit (Anti-Rabbit Dylight 488) 100T
KTD108-EN Universal IF Toolkit (Anti-Mouse Dylight 488) 100T
KTD109-EN Universal IF Toolkit (Anti-Rabbit Dylight 594) 100T
KTD110-EN Universal IF Toolkit (Anti-Mouse Dylight 594) 100T
A21010 HRP, Goat Anti-Mouse IgG 100 μL/500 μL
A21020 HRP, Goat Anti-Rabbit IgG 100 μL/500 μL
A21030 HRP, Rabbit Anti-Goat IgG 100 μL/500 μL
A21040 HRP, Goat Anti-Rat IgG 100 μL/500 μL
A24211 IFKine™ Green Donkey Anti-Mouse IgG 100 µL/500 µL
A24421 IFKine™ Red Donkey Anti-Rabbit IgG 100 µL/500 µL
A24431 IFKine™ Red Donkey Anti-Goat IgG 100 µL/500 µL
A24311 IFKine™ Orange Donkey Anti-Mouse IgG 100 µL/500 µL