Signal pathway investigation

Working protocol of signal pathway investigation

Steps of Western blot experiment：

1. Protein extraction

1.1 The tissue pieces were washed 2-3 times with cold PBS to remove blood stains, cut into small pieces and placed in a homogenization tube. Add 1~2 2 mm small magnetic beads, add 10 times the tissue volume RIPA (add protease inhibitor within a few minutes before use) and place it in the homogenizer, select the program to homogenize thoroughly, about 60s, and cool down intermittently during this period. 

1.2 Take out the homogenized sample tube, ice bath for 30 minutes, and shake it every 5 minutes to ensure complete tissue lysis.

1.3 Centrifuge at 12000 rpm for 10 min, and collect the supernatant, which is the total protein solution.

2. Protein concentration determination: Take the undenatured protein solution and use the BCA protein concentration determination kit to measure the protein concentration.

3. Protein denaturation: Add the protein solution to 5* protein loading buffer at a ratio of 4:1, denature it in a boiling water bath for 15 minutes, and store it in a refrigerator at -20°C for later use.

4. SDS-PAGE electrophoresis cleaning glass plate

a. Cleaning the glass plate

b. Glue filling and sample loading

1. After the glass plate is dry, form a pair of a ground glass plate and a flat glass plate. The gap between the glass plates is a glue-filled gap. Put them into the glue maker, insert a wedge to fix the glass plate, and check if the bottom is aligned to avoid glue leakage.
2. Prepare the separation gel of the required concentration according to the experimental arrangement, and shake it immediately after adding TEMED. Pour the separating glue to an appropriate height. You can try it with a comb before filling the glue. The distance between the comb teeth and the liquid surface of the separating glue is about 5-8mm. Then add pure water slowly and evenly into the gap until it is full, and do not scatter the glue during the process. After about 30 minutes, wait for the separation glue to solidify, then pour off the upper layer of the glue and absorb the remaining water with absorbent paper.
3.  

5% concentrated glue ratio

iv. Prepare 5% concentrated glue according to the above method, and shake it immediately after adding TEMED. Fill the remaining space with concentrated glue and then insert the comb into the concentrated glue. Pay attention that there should be no air bubbles under the comb.

v. After the separation gel has solidified, remove the gel maker and carefully pull out the comb to prepare for electrophoresis.

c. Put the gel maker into the electrophoresis tank and add enough electrophoresis solution to load the sample for electrophoresis. Add the sample to the electrophoresis well and perform electrophoresis. Concentrated gel voltage is 75V, and separation gel uses 120V. After electrophoresis, the electrophoresis can be stopped as soon as the bromophenol blue runs out, and the membrane can be transferred.

4. Transfer film

4.1 Prepare 6 sheets of 7×9cm filter paper and a PVDF membrane of moderate size. PVDF membrane should be activated with methanol before use.

4.2 Put the clamps for transferring membranes, two sponge pads, a glass rod, filter paper and activated PVDF membrane into the basin with transfer fluid.

4.3 Open the clamp to keep the black side level. Put sponge and three layers of filter paper on the mat.

4.4 Carefully peel off the separating glue and place it on the filter paper, and cover the PVDF membrane on the glue without air bubbles. Cover the membrane with three sheets of filter paper and remove air bubbles. Finally, cover with another sponge pad.

4.5 Fast forward. 300mA constant current transfer membrane for half an hour, or 200mA transfer membrane for 1 hour, the time can be adjusted slightly, and the current can be adjusted accordingly. During the transfer process, put the transfer tank in ice water to cool down.

5. immune response

5.1 The transferred membrane was sealed with 5% skimmed milk (0.5% TBST) on a decolorizing shaker at room temperature for 1 hour.

5.2 Dilute the primary antibody (5% skim milk dissolved in TBST, and 5% BSA dissolved in TBST for the phosphorylated protein), and incubate the antibody at 4°C for 3 hours.

5.3 Wash three times with TBST on a decolorizing shaker at room temperature, 5min each time

5.4 The secondary antibody was diluted 3000 times with TBST and incubated for 30 minutes at room temperature, and washed three times with TBST on a decolorizing shaker at room temperature for 5 minutes each time.

6. The ECLA and ECLB reagents were mixed in a centrifuge tube with medium volume in the dark room. Double layer gloves or other transparent films were pasted on the exposure box. The protein side of PVDF film was placed between the two films of the exposure box. Mixed ECL solution was added for full reaction. After 1-2 min, the residual liquid was removed and the upper layer of film was covered to start exposure. The exposed film is developed and fixed with developing and fixing reagents. Adjust the exposure conditions according to different luminous intensity.

7. The film is scanned and archived, PhotoShop sorts and decolors, and the Alpha software processing system analyzes the optical density value of the target zone.

Immunohistochemical procedures：

1. Dewax the paraffin sections to water: sequentially put the sections into xylene Ⅰ 15min- xylene Ⅱ 15min- xylene III 15min- anhydrous ethanol Ⅰ 5min- anhydrous ethanol Ⅱ 5min-85% alcohol 5min-75% alcohol 5min-wash with distilled water.

2. Draw a circle: After the section is dried slightly, draw a circle around the tissue with a histochemical pen (to prevent the antibody from flowing away)

3. Antigen retrieval: Add 0.1% trypsin (0.4% pepsin) dropwise in the histochemistry circle at 37°C for 20 minutes; during this process, prevent excessive evaporation of the buffer and do not dry the tablets. After natural cooling, the slides were placed in PBS (pH 7.4) and washed 3 times with shaking on a decolorizing shaker for 5 minutes each time.

4. Block endogenous peroxidase: put the slices in 3% hydrogen peroxide solution, incubate at room temperature for 25 min in the dark, put the slides in PBS (pH 7.4), shake and wash 3 times on a decolorizing shaker, each time 5min.

5. Serum blocking: Drop 3% BSA in the histochemistry circle to cover the tissue uniformly, and block for 30 minutes at room temperature. (The primary antibody is goat-derived and blocked with rabbit serum, and other sources are blocked with BSA)

6. Add the primary antibody: Gently shake off the blocking solution, add PBS to the slices with a certain proportion of the primary antibody, and place the slices flat in a humid box and incubate overnight at 4°C. (Add a small amount of water in the wet box to prevent the antibody from evaporating)

7. Add secondary antibody: the slides are placed in PBS (PH7.4) and washed 3 times with shaking on a decolorizing shaker, each time for 5 minutes. After the slices were dried slightly, the secondary antibody (HRP-labeled) corresponding to the primary antibody was dropped into the circle to cover the tissue, and incubated at room temperature for 50 minutes.

8. DAB color development: the slides are placed in PBS (PH7.4) and washed 3 times with shaking on a decolorizing shaker, each time for 5 minutes. After the sections were dried slightly, freshly prepared DAB color developing solution was added dropwise to the circle, and the color development time was controlled under the microscope. The positive color was brownish yellow. Rinse the sections with tap water to stop the color development.

9. Counter-stained cell nucleus: hematoxylin is counter-stained for about 3 minutes, washed with tap water, differentiated with hematoxylin differentiation solution for a few seconds, rinsed with tap water, hematoxylin returns to blue, and rinses with running water.

10. Dehydration and mounting: Put the slices in 75% alcohol for 5min-85% alcohol for 5min in sequence-anhydrous ethanol Ⅰ 5min-anhydrous ethanol Ⅱ 5min-xylene Ⅰ 5min for dehydration and transparency, take the slices out of xylene and air dry. Neutral gum mounting film.

11. Microscopic examination, image acquisition and analysis.

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