Experimental principle and precautions of HE staining


HE staining

The full name of HE staining is hematoxylin and eosin staining, which is the most basic and most important pathological staining technique.

HE staining is a routine staining method widely used in pathological diagnosis. A good HE slice is the key to ensure correct pathological diagnosis. The quality of slices can directly affect the timeliness and accuracy of disease diagnosis. As a qualified laboratory technician, being able to produce a high-quality HE-stained section is an important homework that must be mastered.

Experimental principle and precautions of HE staining

1. the principle of nuclear staining:

Hematoxylin is a basic natural dye that stains cell nuclei. The main component of chromatin in the nucleus is DNA. In the double helix structure of DNA, the phosphate groups on the two nucleotide chains are outward, which makes the outside of the DNA double helix negatively charged and acidic, and can easily interact with positively charged cells. The hematoxylin basic fuel is stained by ionic or hydrogen bonding. Hematoxylin is blue in alkaline solution, so cell nuclei are stained blue.

2. the cytoplasmic staining principle:

The main component of the cytoplasm is protein, which is an amphoteric compound. The staining of the cytoplasm is closely related to the pH value. When the pH is adjusted to the isoelectric point of the protein at 4.7-5.0, the cytoplasm does not show electricity to the outside, and it is acidic or alkaline. Dyes are not easy to dye. When the pH is adjusted to 6.7-6.8, the pH value is greater than the isoelectric point of the protein, showing acidic ionization, while the negatively charged anions can be stained by positively charged dyes. At present, the nucleus is also stained, and the nucleus and cytoplasm are difficult to distinguish. Therefore, the pH must be adjusted to below the isoelectric point of the cytoplasm, and acetic acid is added to the dye solution to make the cytoplasm positively charged (cation), which can be dyed by a negatively charged (anion) dye. Eosin Y is a chemically synthesized acid dye that dissociates into negatively charged anions in water, and binds to the positive amino charges (cations) of proteins to stain cytoplasm, cytoplasm, red blood cells, muscle, connective tissue, eosinophilic Red particles, etc. were infected into different degrees of red or pink, in sharp contrast with the blue nuclei.

3. Differentiation:

After staining, some specific solutions are used to remove excessively bound stains from the tissue. This process is called differentiation, and the solution used is called differentiation solution. In HE staining, 0.5% hydrochloric acid and ethanol were used as the differentiation solution, because the acid can destroy the quinoid structure of hematoxylin, so that the tissue and pigment are separated and faded. After hematoxylin staining, it must be differentiated with 0.5% hydrochloric acid and ethanol to remove the hematoxylin dye that is excessively bound to the nucleus and the hematoxylin dye that is adsorbed to the cytoplasm. After eosin staining, the nucleus and cytoplasm can be stained. clear. Therefore, differentiation in HE staining is an extremely critical step.

4. The effect of returning to blue:

After differentiation, hematoxylin is in the red ion state under acidic conditions, showing red, and under alkaline conditions in the blue ion state, showing blue. The tissue sections are red or pink after differentiation with 0.5% hydrochloric acid and ethanol. Therefore, after differentiation, immediately remove the acid on the tissue sections with water to terminate the differentiation, and then use weakly alkaline water (0.2% ammonia water) to stain the nuclei of hematoxylin. It appears blue, and this process is called blue reversion or cyanation. In addition, immersion with tap water can also make the nuclei turn blue, but it takes a long time.

Experimental Materials

Fixative: commonly used 95% ethanol and ice acetone
Hematoxylin staining solution: take by weighing 0.5g of hematoxylin powder, 24g of ammonium alum and dissolve in 70ml of distilled water, then take 31g of NaIO, 5ml of water, then add 30ml of glycerol and 2ml of glacial acetic acid, mix well, filter with filter paper, and set aside.
Eosin staining solution: Weigh 0.5 g of water-soluble eosin staining solution and dissolve it in 100 ml of distilled water.
Diluted hydrochloric acid ethanol solution: prepare 1% hydrochloric acid with 75% ethanol.
A series of concentrations of ethanol, xylene, neutral gum.
Culture flasks, petri dishes, ophthalmic forceps, coverslips, glass slides, microscopes.

Experimental procedure

Sample preparation: For adherent growing cells, trypsin digestion, adjust the cell concentration to about 1 × 105/ml, drop on the coverslip (placed in a 6-well plate), and after culturing for a corresponding time, take out the cell slides, rinse with PBS Wash 3 times.
Sample fixation: 95% ethanol fixation for 20min, PBS wash twice, 1min each time.
Nuclei staining: stained with hematoxylin solution for 2-3 min, and washed with tap water.
Color separation: Observe under a microscope, if the nucleus is too stained, use 1% hydrochloric acid alcohol solution to separate the color for a few seconds, and wash with tap water.
Chromatin cytoplasm: immersed in eosin staining solution for 1 min and washed with tap water.
After air-drying or natural air-drying, the slides were mounted with neutral gum.
If the cells are fixed with 4% paraformaldehyde, the staining time will be prolonged accordingly, 12-15min for hematoxylin staining and 5min for eosin.
Experimental results and precautions

**Experimental results**

The nuclei are blue, and the cytoplasm, muscle fibers, collagen fibers, and erythrocytes are red to varying degrees. Calcium salts and bacteria can be blue or violet blue


1. It is important to adjust the pH when dyeing. If the tissue block is fixed in formalin for a long time, the tissue acidifies and affects the staining of the nuclei. Therefore, rinsing in tap water for a longer time or treating in saturated aqueous lithium carbonate solution for 10-30min can make the nucleus stained darker. When the cytoplasm is not well stained with eosin, 1-2 drops of glacial acetic acid can be added dropwise to the eosin solution.
2. After the sections are stained with hematoxylin, the step of color separation is the key, which should be controlled under the microscope. Generally, it is better if the nucleus is clearly stained (clear) and the cytoplasm is basically colorless. If the color separation time is prolonged too much, the staining will be too light, and the color separation should be done after re-staining.
3. After the slices are dehydrated with alcohol, they may appear white and opaque when they are put into xylene. This indicates that the dehydration is not complete. The slices should be returned to anhydrous alcohol and replaced with alcohol and xylene to achieve complete dehydration and transparency.
4. Do not let the slices dry during the staining process to avoid shrinkage and deformation of the slices, which will affect the neuron morphology.
5. Before the slices are taken out from or into the xylene, the perimeter of the slices should be wiped clean or any excess moisture should be absorbed.
6. In the final sealing, neutral resin should be used to prevent fading in the future. The cover sheet should be larger than the area of ​​the tissue block. If a part leaks out, it will fade soon. The concentration of the resin used should be appropriate, and there should be no air bubbles during resin sealing.