Male Sprague-Dawley rats (8 weeks old; weight 200 ± 20 g) were purchased from Hunan SJA Laboratory Animal Co. Limited (Changsha, China). All procedures were approved by the Second Hospital Animal Care and Use Committee and Xiangya Use Committee, which conformed to the Guide for the Care and Use of Laboratory Animals.
Preparation of gelatin-coated slides
The gelatin solution was prepared by adding 10 g of gelatin (Sigma-Aldrich, catalog number: G7041) in 1000 ml of double-distilled water (DW), which was constantly stirred and heated until the gelatin dissolved. One gram of chromium potassium sulfate [CrK (SO4) 2 · 12H2O; Sinopharm; catalog number: 20015260] was added to the solution and stirred continuously.
Then we filter the solution with filter paper. Immerse the clean slides on the rack in the solution for 10 s avoiding air bubbles and then place it in the oven (65 ° C) overnight. The slides could be used in a month. The gelatin prevents sections of the brain, which are primarily made up of fat and water, from sticking to the slides. Chromium potassium sulfate provides positive ions for the slide. Therefore, the brain tissue could adhere firmly to the slides.
Note: It is important to use gel-coated slides, otherwise the brain section will fall off the slide during the staining procedure. Gelatin-coated slides should be used within 3 months of preparation; otherwise, the section may crack after mounting on the slide while it dries.
- Preparation of the impregnation solution
- Three stock solutions are prepared as follows:
- Solution A: a 5% solution of potassium dichromate (K2Cr2O7; Sinopharm, catalog number: 10016618) in 100 ml DW
- Solution B: a 5% solution of mercury chloride (HgCl2; Sinopharm, catalog number: 10013616) in 100 ml DW
- Solution C: a 5% solution of potassium chromate (K2CrO4; Sinopharm, catalog number: 10016418) in 80 ml DW
These solutions must be dissolved and shaken. Heating is necessary when preparing solution B. The stock solution should be kept in the dark for a few months. Mix 5 vol. parts of solution A, 5 vol. parts of solution B, 4 vol. parts of solution C and 10 vol. of DW stirring them. After sufficiently mixing the solutions, the working solution should be kept in the dark for at least 24 h, during which time reddish precipitates form. Remove the precipitates with filter paper.
A total of 20 ml of working solution is required for each rat brain sample. The volume of the working solution depends on the number of tissues, and the working solution can be used up to one week after preparation. Metallic instruments should be avoided during preparation. Tissues treated with the Golgi-Cox solution should be protected from exposure to light whenever possible.
The cryoprotective solution is essential to protect cryosections, including preservation of morphology and protection of frozen tissue from artifacts. One hundred milliliters of a tissue protection solution (solution D) are prepared by dissolving the following components: 20 g of sucrose (C12H22O11; Sinopharm, catalog number: 10021463), 15 ml of glycerol (C3H8O3; Sinopharm, catalog number: 10010618), and the volume is then adjusted to 100 ml with DW. One hundred milliliters of cryoprotectant solution is sufficient for two rat brain samples.
For the staining step, solutions E and F are needed. Solution E (20% ammonia solution) can be prepared by adding 150 ml of ammonia (NH4OH, 25.0% -28.0%; Sinopharm, catalog number: 10002108) in 50 ml of DW. Solution F (1% sodium thiosulfate solution) is prepared by dissolving 3.14 g of sodium thiosulfate (Na2S2O3 · 5H2O; Sinopharm, catalog number: 10021218) in 200 ml of DW.
Note: the above-mentioned solutions are toxic and harmful, perform the experiment under a smoking hood and wear protective gloves.