HE staining
Hematoxylin-eosin staining, referred to as HE staining, one of the commonly used staining methods in paraffin sectioning techniques. Hematoxylin dyeing solution is alkaline, mainly causing chromatin in the nucleus and ribosome in the cytoplasm to be purple-blue; eosin is an acid dye, which mainly causes redness in the cytoplasm and extracellular matrix. HE staining is the most basic and widely used technical method in the teaching and research of histology, embryology and pathology.
1 Scope of application
Histology, embryology, pathology teaching and research.
2 principle
The property of being easily colored by an alkaline or acid dye is called basophilia and acidophilia; and the phenomenon that the affinity between the basic dye and the acid dye is weak is called neutrophilia.
Proteins in tissues constitute a wide variety of amino acids, and they have different isoelectric points. In the ordinary staining method, the pH of the staining solution is about pH6, and the acidic substances in the cells such as the chromatin of the nucleus, the rough endoplasmic reticulum in the gland cells and nerve cells, and the hyaline cartilage matrix are all dyed with basic dyes. Called basophilic. Other proteins in the cytoplasm, such as hemoglobin in red blood cells, particles of eosinophils, and collagen fibers and muscle fibers, are stained with acid dyes, which are called acidophilic types. If the pH of the dyeing solution is changed and the pH is increased, the substance originally stained with the acid dye may become basophilic; when the pH is lowered, the substance originally stained with the basic dye may become eosinophilic. Therefore, the pH of the staining solution can affect the reaction of the dyeing.
The phosphate group on both strands of deoxyribonucleic acid (DNA) is outwardly, negatively charged, and acidic, and is easily dyed by ionic bonding with a positively charged hematoxylin basic dye. Hematoxylin is blue in an alkaline solution, so the nucleus is stained blue. Eosin Y is a chemically synthesized acid dye that dissociates into a negatively charged anion in water and binds to the amino-positively charged cation of the protein to stain the cytoplasm. Cytoplasm, red blood cells, muscle, connective tissue, eosin particles It is dyed to varying degrees of red or pink, in stark contrast to the blue nuclei. Eosin is a good dye for cytoplasm.
Due to the different affinity of the different components of tissue or cells for hematoxylin and the different dyeing properties. After hematoxylin staining, the nucleus and calcium salt mucus are blue, and can be differentiated with hydrochloric acid alcohol and weakly alkaline solution. If the treatment is suitable, the cells can be clearly dark blue, and other components such as cytoplasm are decolored. The cytosolic dye is used to dye the cytoplasm, so that the various components of the cytoplasm show different shades of pink. Therefore, the general morphological and structural characteristics of various tissues or cell components and lesions can be displayed.
3 reagents and instruments
A: 0.5~1% eosin alcohol solution:
Weigh E-Y 0.5 to 1 g, add a small amount of distilled water to dissolve, and then add glacial acetic acid until the paste. Filtered with filter paper, the filter residue is baked in an oven, and dissolved in 100 ml of 95% alcohol (ie, industrial alcohol, if not afraid of waste, with anhydrous ethanol).
B: Hematoxylin dye solution: (Form 3000 ml, can be reduced by the ratio)
Hematoxylin 6 g
Anhydrous ethanol 100 ml
Potassium aluminum sulfate 150 g
Distilled water 2000 ml
Sodium iodate 1.2 g
Glacial acetic acid 120 ml
Glycerin 900 ml
The preparation method comprises the following steps : dissolving hematoxylin in anhydrous ethanol, dissolving potassium aluminum sulfate in distilled water, dissolving and then pouring the glycerin together, and finally adding glacial acetic acid and sodium iodate.
C: 1% hydrochloric acid alcohol differentiation solution: 1 ml of concentrated hydrochloric acid can be added to 99 ml of 70% alcohol. [
4 steps
Conventional he staining step
(1) xylene (I) 15 min
(2) xylene (II) 15 min
(3) xylene: absolute ethanol = 1:1 2 min
(4) 100% ethanol (I) 5 min
(5) 100% ethanol (II) 5 min
(6) 80% ethanol 5min
(7) distilled water 5 min
(8) Sumu semen staining 5 min
(9) Wash the water slightly to the hematoxylin 1-3 s
(10) 1% hydrochloric acid ethanol 1-3 s
(11) Wash it slightly 10-30 s
(12) distilled water wash 1-2 s
(13) 0.5% eosin staining 1-3 min
(14) Washing with distilled water 1-2 s
(15) 80% ethanol wash slightly 1-2 s
(16) 95% ethanol (I) 2-3 s
(17) 95% ethanol (II) 3-5 s
(18) Anhydrous ethanol 5-10 min
(19) Carbonic acid xylene 5-10 min
(20) xylene (I) 2 min
(21) xylene (II) 2 min
(22) xylene (III) 2 min
(23) Neutral gum sealing Note: 1 (11), (12) can be omitted, (13) step watering time should be extended to 20-30min.
2 Step (21) If no carbonic acid xylene is used, anhydrous ethanol can be used instead.
Precautions
The slice is dehydrated and transparent. After being stained by HE, it should be thoroughly dehydrated and transparent before it can be covered with a neutral gum. If the dehydration is not complete, the film will be white mist after the film is closed, and the observation under the microscope is unclear and easily faded. Slicing 1-2 grade anhydrous ethanol,
Dehydration can also be carried out using xylene carbonate. Carbolic acid has a strong dehydration ability, but the slice can be decolored for a long time, so it is necessary to pass a plurality of xylenes to completely remove the rock carbonic acid.
5 judgment editor
Experimental result
The nucleus was stained with hematoxylin by a bright blue color, the cartilage matrix and calcium salt particles were dark blue, and the mucus was grayish blue. The cytoplasm is stained with eosin to a different shade of pink to pink, and the eosinophilic granules in the cytoplasm are bright red with a strong reflection. The collagen fibers are light pink, the elastic fibers are bright pink, the red blood cells are orange-red, and the protein liquid is pink. [1]
The coloration is related to the type of tissue or cell, and also changes with its life cycle and pathological changes. For example, cells in the neonatal period have a lighter or mildly alkalophilic effect on eosin, and eosinophilic staining occurs when they age or undergo degenerative changes. When the collagen fibers are aging and appear to be hyalinous, the color of the eosin becomes darker and darker.
HE dyeing evaluation criteria:
(1) The slice is complete, the thickness is 4-6 microns, the thickness is uniform, and there are no wrinkles and no knife marks;
(2) The dyed nucleoplasm is distinct, the red and blue are moderate, transparent and clean, and the seal is beautiful. [3]
Hard Capsule,Acne Remove Capsule,Skin Lighten Capsule,Anti-Inflammatory Capsule
Guangzhou Etechange Biological Engineering Co.,Ltd. , https://www.etechange.com