Cell climbing sheet manufacturing method Preparation for climbing 1. The general cover slip can be used for the climbing piece, and the special climbing piece can also be used; 2. Apply cover slips, which can be cut to the right size according to your needs, so that you can put them in 6-well, 12-well or 24-well plates. You can use the former nurse infusion to open the small sand wheel of the glass ampoule. Or the small sand wheel of the stomatology department, after a light stroke, it will be separated with a little force. If you inoculate a large dish, I generally do not cut the coverslip, directly clean it, put it into the culture dish, and then cut it when dyeing, which not only ensures cell homogeneity, but also can do several indicators at the same time. . 3. The cut pieces are immersed in concentrated sulfuric acid and left overnight. The next day, rinse with tap water for 20 times, then soak in anhydrous wine for 6 hours, then rinse with 3 times of steamed water for 3 times. It is considered that the main purpose is to rinse the acid clean. If it is not clean, the cell wall is not good. It is dried in a lunch box or a glass culture dish and then subjected to autoclaving. 4. After high pressure, take it out and put it in the oven and bake it for later use. After baking, it can be placed in a clean bench. 5. With regard to polylysine, many people use this slide to make the cells more firmly bonded to the slide. However, I have never used too much polylysine when doing climbing. The cell adhesion is still very good, and I have never taken off the film after doing the subsequent immunocytochemical staining, TUNEL and other apoptosis detection, immunofluorescence and so on. [Cell crawler] 1. After trypsinizing the cells, resuspend the cells in complete medium. 2. When adding cells, according to the size of the slide, first place a small amount of medium in the position where the slides are prepared in each well. The purpose is to bond the slide and the culture dish to the tension of the culture medium, and then put the glass. Tablets prevent the slide from undulating when the cell suspension is added, resulting in a double-layered cell patch. Pay attention to aseptic operation throughout the process. 3. According to your needs, select the appropriate cell density into the culture plate. 4. After the cells are attached, you can go to the supernatant and add the drug-containing medium. 5. According to the experimental design, take a slide after a certain time. When the slide is taken, because the slide is tightly combined with the bottom of the culture dish, the tension is relatively large. I usually put a small hook on the back of the needle tip of the syringe needle, so that the climber is gently hooked up and removed with a small forceps. If you want to do experiments such as 24h, 48h, 72h, etc., use the alcohol lamp flame to burn the needle and tweezers when removing the required number of climbing pieces. The last taken out can be continued with the cultivation. [Fixation of cell slides] After the cell slides are taken out, they are usually washed twice with PBS and then fixed. Of course, depending on the purpose of the study, PBS washing is not a must, such as avoiding washing when TUNEL staining for apoptosis, because apoptotic cells may fall off during washing. In addition, when saving cell slides, I usually use a petri dish or plate, first put a layer of tissue paper on the bottom of the dish, and then put the piece to climb, so that it is convenient to take the film during the experiment. Then cover the lid and mark it on the lid. To avoid confusion, use a clear tape to join the lid to the dish. It is generally no problem to store 2-3 months of film at -20 °C for immunohistochemistry. The following are commonly used fixatives 1. Cold acetone can be used for general immunohistochemical staining. Put the pure acetone into the refrigerator at -20 °C and then use the cold acetone (safe, acetone at this temperature will not be solid) cell climber, take PBS and wash it twice, then add cold acetone at -20 °C ( Acetone is highly volatile, and the vessel containing acetone and climbing tablets should be tightly sealed to prevent it from evaporating for 10 minutes. After taking out, it was dried at room temperature and then stored at -20 ° C. 2. Paraformaldehyde (usually 4%): can be used for immunoelectron microscopy; it can also be used for immunofluorescence and TUNEL staining. It mainly detects some delicate antigens in tissues, especially cell surface antigens, such as various lymphatic differentiation syndrome (CD), major histocompatibility antigens, etc. After 15 minutes at room temperature, the surface liquid is blown dry, into - Store at 20 ° C. 3. 95% ethanol, which can be used for immunohistochemistry. Advantages: strong penetrability and good antigenic preservation. Disadvantages: However, alcohols have poor preservation effect on low molecular weight proteins, peptides and cytoplasmic proteins, so that the effect of protein denaturation is light, and can be redissolved after fixation. During the dyeing process, the incubation time is long, the antigen can be lost and the reaction intensity is weakened. After fixing for 15 minutes at room temperature, dry the surface liquid and store at -20 °C. 4. Formaldehyde (formalin) has the widest application advantages: good morphological structure preservation and strong penetrability. Disadvantages: Formaldehyde can be oxidized to formic acid for a long time, so that the pH of the solution is lowered, which affects the dyeing; The grid structure may partially or completely mask certain antigenic determinants from being sufficiently exposed. It can cause false negative staining results. After fixing at room temperature for 15 minutes, the surface liquid was blown dry and stored at -20 °C. Concealed Slides,Push Open Undermount Slide,Concealed Drawer Slides,Concealed Undermount Drawer Slides JOKER UNO LIMITED , https://www.jokeruno.com
Zhengzhou Weier-cell climbing sheet manufacturing method