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H&E Staining Method


H&E Staining Method

 

As one of the most basic methods of pathology laboratory, histopathological biopsy technique has high value of use and is the most basic and common morphological testing technique for clinical surgical pathology. Usually pathological section mainly includes the paraffin section and H&E staining, roughly the operating procedure include tissue fixation, embedding, section, floating, dehydration, transparent and mounting. The quality of histopathological biopsy influence pathological diagnosis result. H&E staining is basic and important job, the staining quality affect pathologist’s diagnosis. 

 

The materials should be as fresh as possible to prevent the necrosis of organ tissue, it should be very fast.A sharp blade or scissors is used for the purpose of avoiding the rupture of the tissue. The thickness of the tissue block is selected, and the size should be moderate. If the tissue block is too thick or too large, it will be prone to distortion and distortion in fixed dehydration.The materials should also be representative and reflect the basic structure of the organization;In addition, it is necessary to collect the tissue of the healthy part and the inferior part of the lesion, so as to be able to see the various pathological changes clearly through the comparison of the two.Normally, the fixed liquid that is configured should be used in time, should be stored in the shade, avoid light preservation, so as to avoid chemical changes and lose their fixation.Be timely in order to ensure that the cells are similar to normal living conditions.The fixed fluid should be sufficient, generally about 25 times the material fast, for the material with more water content, should replace 2-3 times new fluid, avoid the occurrence of corruption.

 

The material is fixed, should be kept in the sealed container and labeled outside the container, indicating the source of the material, the fixed solution and the date and other main information.Material after fixed, in addition to ethanol and other fixed fluid must be rinsed clean, otherwise remain fixed liquid materials within the organization may produce crystallization or precipitation, and bad for staining.

 

In the pathological tissue section, the method of embedding wax embedding was adopted, and the wax temperature should remain constant. The embedding operation should be done quickly, as far as possible in the shortest time to complete the process of paraffin wax, avoid causing the tissue to become brittle, shrink or harden, etc.Under normal circumstances, the embedding operations can be finished in the constant temperature box, in order to avoid tissue materials due to the change in temperature on separation of paraffin wax, organize material section should be placed upside down, and USES the tweezers gently upon material with temperature, so that it is flat and level, can take out the spare for paraffin solidified completely.When slicing, make sure that the sliced knife and blade are used, otherwise the slice fracture and incomplete will be caused.When slicing, pay attention to the inclination of the blade to be appropriate, the Angle too small to cause wrinkles, too large to roll up the section;In general, the thickness of a slice of 5um is selected. If the slice is too thick, it may result in double layer cell phenomenon, which is not conducive to observation and pathological diagnosis.In the patch operation, a small tweezers shall be used to pick out the paraffin strips that have been cut off, and put them above the water surface of the thermostatic water bath at 37 degrees.After the display, the microscope slides are marked with a temperature of about 14h in a 55-degree oven to prevent the film from taking off.

 

Since most of the staining solution is aqueous solution, the wax should be removed before staining, and the process of gradual rehydration and dewaxing is the reverse of the process.However, it should be noted that because of the thin wax, the time required is much shorter. After removing the wax, the slices are placed in the suwood essence for about 8 minutes. The staining time should be determined according to the room temperature and the degree of staining agent.When room temperature is high, promote staining, should shorten the staining time, the winter room temperature should be placed in the incubator staining.Rinse water with water after staining must not be too large, do not run straight, and should be examined by microscope at any time until the color of the section is blue.Pathological biopsy after staining, dehydration, transparent, should immediately with cementing agent to be sealed, sealing the section should have a small amount of xylene, otherwise it may appear pigment, in the process of cover glass placed, if too much glue solution, should be dry after use the blade scrape, dip in with gauze wipe residual gum with xylene.In order to avoid the indoor temperature, the gas from the nose should be exposed to the rubber section, and the sealing action should be as soon as possible.If take off a piece after dyeing, may be because of slicing, when attached to the water temperature is low, tissue dehydration is not completely lead to paraffin slice thickness caused by inadequate, flushing water too hard, or the organization itself is easy to fall off, such as blood clots.


Pathological biopsy is done, need to put a period of time stay dry gum to archive, otherwise there may be sticking phenomenon, archive section should be placed in dry cool and dark place, avoid fade.If there is a discoloration, it may be due to dehydration, transparency and other procedures. The cover glass is smaller, the section is exposed to the sun, and the sealing agent used is not neutral.


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