Source: Practical Experimental Techniques in Neurobiology, Fourth Military Medical University Press

Operation method

basic program

Materials and Instruments

Laboratory animals
Anhydrous ethanol, xylene, alcohol, glycerin, saline, etc.
Perfusion bottles, needles, scissors, regents, clamps, tissue forceps, bone-biting forceps, etc.

Move

1 Anesthetize the animal with 1% pentobarbital sodium (80 mg/kg), cut open the thoracic cavity along the sternal styloid, cut open the pericardium, expose the heart, cut open the left ventricle at an angle of 30°-45° to the apex of the heart, then cut open the right auricle, and insert a perfusion needle from the left ventricle into the aorta, and fix the needle (Fig. 2-I), and then fix the infusion. Anesthetize the animal with 1% pentobarbital sodium (80mg/kg), cut open the thoracic cavity along the sternal stalk, cut open the pericardium, expose the heart, cut open the left ventricle at an angle of 30°-45° to the apex of the heart, cut open the right auricle, insert the perfusion needle from the left ventricle into the aorta, and fix the needle (Fig. 2-I), and then flush the blood with saline quickly, and inject the fixative solution into the heart quickly and slowly to fix the heart by perfusion for 1.5 h. (Table 2-2), if the fixation is unsatisfactory, the material can be taken out and then fixed for 2-4h (4°C), then the heart is fixed by perfusion for 1.5h (4°C). If the fixation is unsatisfactory, the material can be removed and fixed for 2-4h (4 ℃).

2 take animals after perfusion fixation must be taken in a timely manner, the tissue taken by the site, size, shape according to the experimental needs to follow the principles of gentle movement, avoid extrusion, sectioning flat, the purpose of the tissue intact and so on. Remove the desired tissues and soak them in 20% sucrose solution (4 ℃) until the tissues are completely dehydrated and sink to the bottom.

Caveat

(1) Irrigation should be rapid and there should not be too much saline. The perfusion of fixative should be fast and then slow for adequate fixation.(2) The total most of the fixation solution is usually about 2 times of the animal's whole body blood.(3)The volume of fixation liquid is about 20 times of the tissue when immersion fixation.(4) Take the material need to be careful, do not tear the tissue, or too much force, resulting in tissue damage.(5) Sodium pentobarbital, picric acid and other reagents are poisonous and anesthetic drugs and need to be managed by special personnel.

Common Problems

Other reagents: ① 1% sodium pentobarbital solution (100ml) weigh 1g of sodium pentobarbital, 100ml of saline, fully dissolved (placed at 4 ℃); ② 0.1mol / L PB buffer (1000ml) were weighed _{KH2P04 13} .6g, NaOH 3.20g, add double-distilled water to 1000ml, adjust the pH to 7.4; ③ 4% paraformaldehyde solution (1000ml) were weighed NaOH 3.20g, paraformaldehyde 40g, _{KH2P04 16} .80g, distilled water to dissolve, filtered and fixed, adjust the pH to 7.4 (need to use a stirrer to stir, pay attention to the order of addition, the first completely dissolved and then add the next one, no need to heat can be quickly dissolved); ④ paraformaldehyde - glutaraldehyde -Picric acid mixed fixative;

(1) The choice of fixative is important, methanol is not ideal for antigen preservation, ethanol is not suitable for T and B cell antigen fixation.

(2) Perfusion fixation is a critical first step for good immunohistochemistry. The physiological saline should not be too much to prevent too much causing tissue edema, and the animals should be taken in time (within 2h) after the end of perfusion fixation to avoid prolonged shelving. If the animal is not well fixed by perfusion, it can be post-fixed for 2-4h (4℃) after taking the material.

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