In situ staining for β-galactosidase can be applied (1) to determine whether the guide DNA has been integrated into the host cell and (2) to trace cell-cell interactions.

Operation method

In Situ Staining Technique

Principle

Beta-galactosidase is a commonly used reporter gene molecule that can be used to detect beta-galactosidase expression by in situ staining under a normal light microscope.

Materials and Instruments

Transfection of cells with β-gal expression plasmid
D-PBSA staining solution
6-well culture plate Thermostat Inverted microscope

Move

I. Materials
Non-sterilized items

1. D-PBSA

2. Substrate: X-gal (nvitrogen), 20 mg/mL in dimethylformamide, stored in polypropylene tubes at -20°C, protected from light, for up to 6 months.

3. fixative: 1.8% formaldehyde and 0.05% glutaraldehyde, both prepared in PBSA solution; 85 mL of water, 10 mL of 10x D-PBSA, 5 mL of formalin (37% formaldehyde solution), and 0.2 mL of glutaraldehyde (25% solution) are mixed together to form a fixative, stored at 4℃.

4. Staining solution: 5 mmol/L potassium ferricyanide, 5 mmol/L potassium ferrocyanide, 2 mmol/L MgCl₂, prepared in D-PBSA, stored at 4℃.

5. Substrate/staining solution: 1 mg/mLX-gal, prepared in staining solution, ready to use.

6. D-PBSA solution containing 10% formaldehyde

7. Transfect the expression plasmid of β-gal as a reporter gene (e.g. pcMV β-gal).

Second, the operation steps:
1. Wash the cells with 2 mL of D-PBSA (e.g., in a 6-well culture plate).

2. Fix the cells with 1 mL of fixation solution for 5 min at room temperature.

3. Wash the cells twice with 2 mL of D-PBSA.

4. Add 1 mL of substrate/staining solution into each well and incubate at 37℃ for 2 h.

5. Rinse each well with 2 mL of D-PBSA solution, observe the cells under an inverted microscope and count the blue-stained (β-gal positive) cells.

6. Storage of culture plates. Add 1 mL of buffer salt solution containing 10% formaldehyde to each well, fix it at room temperature for 10 min, then wash the cells with buffer salt solution and store the buffer salt solution at 4℃.

Caveat

1. β-Gal Fixative has certain corrosiveness and odor, please pay attention to protection when handling.

2. β-Galactosidase staining reaction is dependent on specific pH conditions and cannot be performed in a carbon dioxide incubator. Higher concentrations of carbon dioxide in a carbon dioxide incubator used for cell culture may affect the pH of the staining solution and cause staining failure.

3. Polypropylene or glass containers should be used to prepare the staining solution, and polystyrene containers should not be used. However, staining can be done in polystyrene containers, for example, ordinary 6-well plates can be used as staining containers.

Common Problems

1. A staining assay for senescent cells or tissues based on the up-regulation of SA-β-Gal (senescence-associated β-galactosidase) activity during senescence. 2.

2. For tissue sections: (1) For paraffin sections, dewaxing and hydration are performed in the usual way. For frozen sections, directly follow the steps below. (2) Add an appropriate volume of β-galactosidase staining fixative to sufficiently cover the tissue, and fix for not less than 15 minutes at room temperature. (3) Wash the tissues with PBS immersion 3 times, each time not less than 5 minutes. (4) Observe under an ordinary light microscope. If it can not be observed in time, plus the sealing solution can be stored for a longer period of time after sealing at 4℃.

Source Animal Cell Culture: A Guide to Basic Techniques (Fifth Edition)

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