Animal tissue sampling is the first step for subsequent pathological staining or molecular detection experiments, and it is also a very critical step. After the materials are collected, they must be effectively fixed before paraffin embedding, sectioning, and subsequent experiments. Now we will introduce tissue sampling, selection of sectioning direction and common pathological special staining.

Figure 1. Mouse brain tissue processing and paraffin embedding flow chart. (Smart A, et al.; 2023)

Overall Considerations for Collecting Materials

1. During the sampling process, instruments must not cause physical damage to the specimen;
2. Remove fresh specimens and fix them immediately. If there is blood on the surface of the tissue, rinse the blood with PBS or saline first and then fix it;
3. When collecting materials for pathological experiments, the thickness of the tissue should not exceed 0.5cm, and the length and width should not exceed 1.5cm;
4. When fixing, the container should not be too small, the specimen should not be squeezed by the container, and the fixative should account for more than two-thirds of the volume;
5. Tissues that are easy to float, such as lungs, should be covered with gauze or cotton wool, and tissues with cavities, such as stomach, bladder, etc., should be cut and fixed;
6. When fixing, the fixative should be poured in first and then the tissue;
7. Selection of fixatives for special tissues and organs: such as eyeball-specific fixatives and fat-specific fixatives;

Brain (sampling, embedding, special staining)

Draw Materials

It is recommended that the animal should be perfused with 4% paraformaldehyde fixative before stripping off the brain tissue.

1. Brain tissue is soft and cell components are difficult to retain;
2. After fixation, brain tissue damage can be reduced during brain removal operations;
3. Blood is present in the brain tissue, and subsequent staining or IHC can remove background effects;

Note: Animal tissues that have been perfused with paraformaldehyde cannot be used to extract RNA, protein, etc. for PCR, WB, and ELISA biochemical testing.

Embedding Section Direction

Coronal plane-observe the typical structures of the prefrontal cortex or striatum or hippocampus, thalamus, hypothalamus or the largest surface of hippocampus, substantia nigra, etc. respectively.

Sagittal plane-simultaneously observe the olfactory bulb, cortex, striatum, hippocampus, cerebellum, etc. This section is recommended to observe the distribution of tumor cells in brain tissue.

Horizontal plane-can simultaneously observe the olfactory bulb, cortex, striatum, hippocampus, brainstem, cerebellum, etc.

Special staining of the brain

• HE Staining

a) Purpose and application of staining--observation of pathological changes in brain tissue

b) Histopathological description - the brain tissue in the infarct area is loose and edematous, the capillaries are dilated and congested, neuronal vacuolar degeneration, some neurons are pyknotic and triangular, the number of Nissl bodies is reduced, and glial cells proliferate.

c) Specimen preparation method--fixed with 4% paraformaldehyde, embedded in paraffin and sectioned

• TTC Staining

a) Purpose and application of staining--to display the location of ischemic infarction in brain tissue and to measure the size of the infarct area are pyknotic and triangular, the number of Nissl bodies is reduced, and glial cells proliferate.

b) Histopathological description - bright green fluorescence in necrotic neurons are pyknotic and triangular, the number of Nissl bodies is reduced, and glial cells proliferate.

c) Specimen preparation method--fixed with 4% paraformaldehyde, embedded in paraffin and sectioned are pyknotic and triangular, the number of Nissl bodies is reduced, and glial cells proliferate.

• FJB Fluorescent Staining

a) Purpose and application of staining--observation of necrotic neurons in brain tissue

b) Histopathological description - normal tissue is red and infarcted area is pale.

c) Specimen preparation method - fresh tissue or -800℃ frozen tissue cut into 2mm tissue blocks for staining

• Nissl Stain

a) Purpose and application of staining-to display Nissl bodies in neurons and reflect the status of neurons

b) Histopathological description - Nissl bodies in neurons are dark blue granules, nuclei are light blue, and the number of Nissl bodies in the infarct area is reduced.

c) Specimen preparation method--fixed with 4% paraformaldehyde, embedded in paraffin and sectioned

• Silver Dyed

a) Purpose and application of staining--to display the condition of neuron fibers

b) Histopathological description—neuronal axons, intraneuronal fibrils, neurofibrillary tangles, age spots and dendrites are black with a golden background

c) Specimen preparation method--fixed with 4% paraformaldehyde, embedded in paraffin and sectioned

Related Products

1. Smart A, et al.; Protocol for tissue processing and paraffin embedding of mouse brains following ex vivo MRI. STAR Protoc. 2023, 4(4):102681.

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