JRD250 immunofluorescence experiment

Cellular immunofluorescence experiment dyeing

: : 1. Material preparation 1) cover slide. Slide
cover slide water is soaked in 75% Ethanol solution Ultrasonic acid Tide water washing anhydrous ethanol soaked drying 2) reagent a. Fixed liquid: 4% formaldehyde, 4% polymethylene (PBS, 72 degree dissolution) b. Light membrane: 0.2% or 0.3% Triton X-100, methanol + acetone (V: v u003d 1: 1) c. Closed liquid: 5% BSA
More reagents De Further methanol + Preparation of PBS
d. Anti-: Rabit-Anti-HA (1: 500), Mouse-Anti-Myc (1: 500),
mouse-anti-flag (1: 1500)
e. Secondary:
Goat-Anti-Rabbit Fitc (1: 500), Goat-Anti-Rabbit-Mouse Texas (1: 500)
Anti-secondary anti-sealing is diluted [ 123] f. Hoechst 5ug / ml PBS Dilution
g. 90% glycerol is dissolved in PBS
h. Nail polish
2. Slide
In order to make some cells before cell culture The wall performance of, for example, 293, 293T) is improved to treat the cover slide. Commonly used treatment methods are:
A: Gelton treatment:
1. Preparation 2% gel aqueous solution, high pressure Sterilization (also contribute to the dissolution of gel)
2. Put the appropriate amount of cover slide (generally no more than 40 pieces) into 10 cm cell culture dish
3. Add 10 ml of 2% gel solution On the sputum shaker, SHAKE 1 hour at room temperature.
4. Above 200 ul of 25% isoprealdehyde to 10 mL of newly prepared 1XPBs
5. Sucking gel solution, add newly prepared isoprealdehyde solution, and shake at room temperature for 15 minutes.
6. Wash 5 times with PBS, 5 minutes each time.
7. Place the coverslide on a 30mm disc or a six empty plate, UV irradiation for 1 hour, standby.
B: Multi-lysine treatment
1. Place the wash sand slide in 40 u0026 mu; g / ml polylysine treatment solution, soak for about 1 hour at room temperature
2. Wash the tap water for 1 hour, then soaked 3 times with distilled water, 5 minutes each time.
3 UV irradiation 3 hours, spare.
3. Cell culture
According to different growth rates of cells, the density of the cells is adjusted, and in general, the density of 1 x 106
is preferably 1 x 106
4. Transfection [ 123] In order to detect the cellular positioning of a protein molecule or observed that the two protein molecules in the cellular positioning, a gene is often transfected into the cell, and there is a commonly used PEI, calcium phosphate transfection method.
5. Dyeing
1) After transfection 24 h, the culture solution was sued, PBS washed once
2). Fixed: 4% formaldehyde or 4% polymethylene, 20min, PBS wash secondary [ 123] 3). Percubic membrane: a. 0.2% or 0.3% Triton X-100, RT, 10min, PBS wash secondary
b. Methanol + acetone (V: v u003d 1: 1), -20 u0026 deg; 20 min, PBS wash three times
4). Closed: Closed 700 ul, RT, 60min (5). One-resistance: 100 ul, RT, 1HR or 37 u0026 Deg; 45min 5 min / Time
6). Second Anti-Anti-: 100 ul, RT, 1HR or 37 u0026 Deg; 45min, PBS wash 2 times, 5min / Time
7). Dye Nuclear: Hoechst, 200 ul 4 times, 5 min / Time
8). Sealed: 9 ul 90% glycerin. Do not leave with bubbles.
6. Fluorescence microscope observation