Immunostaining Antibody Eluent

 

Product Name	Cat. No.	Spec..
Immunostaining Antibody Eluent	G1266	30 mL

 

 

This product is employed for the elution of primary and secondary antibodies during immunofluorescence (IF), immunohistochemistry (IHC), and immunocytochemistry (ICC) immunostaining of tissues and cells. This Immunostaining antibody eluent has the advantages of simple operation, mild conditions and strong elution effect. It can completely eluate primary and secondary antibodies without damaging the morphology and structure of the sample. It is especially suitable for the antibody elusion in the TSA immunofluorescence multi-label experiment of paraffin sections, frozen sections, cell slides, cell smear, etc. of brain, bone, skin, aortic valve, etc. The non-covalently bound primary antibody and the target protein, the second antibody and the primary antibody were interrupted and eluted to remove the influence on subsequent antibody labeling.

 

 

2~8℃ storage and transport, valid for 6 months.

 

 

Component Number	Component	G1266-30 mL
G1266	Immunostaining antibody eluent	30 mL
Manual		1 pc

 

 

This product is ready-to-use and does not require dilution. Before use, remove the antibody eluent from the refrigerator and restore it to room temperature before adding a small amount of antibody eluent to cover the sample, incubate at room temperature for 5min, then discard the eluent, add enough eluent again to completely cover the tissue, incubate at 37°C for 30min, place the slide in TBST and wash it on a shaking table for 3 times, for 5min each time.

The application of the antibody eluent is described in detail with the example of TSA fluorescent multi-labeling in paraffin sections:

1. Tissue sections were dewaxed to water: Sequentially, the sections were put into Eco-friendly dewaxing solution I (G1128) for 10 min - Eco-friendly dewaxing solution II (G1128) for 10 min - Eco-friendly dewaxing solution III (G1128) for 10 min - anhydrous ethanol Ⅰ for 5 min - anhydrous ethanol Ⅱ for 5 min - anhydrous ethanol Ⅲ for 5 min - and washed with distilled water;

2. Antigen repair: According to the tissue type and antibody type, select the corresponding antigen repair solution (G1219,pH6.0; G1207,pH8.0; G1218,pH9.0) and repair mode for antigen repair. The general repair method is antigen repair instrument (Servicebio forthcoming products), microwave, high pressure and other high-temperature repairs. During this process, the repair solution should be prevented from excessive evaporation and should not be dried. After natural cooling, the slide was placed in PBS (G2156) and washed by shaking on the pendulum table (SYC-Z100) for 3 times, 5 min each time.

3. Pap Pen drawing circle and seal with hydrogen peroxide: After the sections are slightly dried, draw circles around the tissues with a Pap pen (G6100), which is generally about 3 mm away from the tissues. After the circles are finished, wash them with pure water (G4701) or PBS (G2156) after the circles are dried, and then put the sections into 3% hydrogen peroxide solution and incubate at room temperature for 25 min in order to block endogenous peroxidase. After closure, the slide was placed in PBS (G2156) and washed by shaking on pendulum table (SYC-Z100) for 3 times, 5 min each time.

4. Serum blocking: After the section is slightly dried, 3%BSA or serum is added to the tissue and sealed at room temperature for 30 min, and the blocking reagent is determined according to the primary and secondary antibody species.

5. Add the first primary antibody: Gently shake off the sealing solution, add the primary antibody in a certain proportion to the slice, put the slice flat in a wet box (SIB-20F), incubate at 4°C overnight. (Add a small amount of water to the bottom of the wet box to prevent the antibody from evaporating)

6. Add the corresponding HRP labeled secondary antibody: The slide was placed in PBS (G2156) and washed on the pendulum shaker (SYC-Z100) for 3 times, 5 min each time. After the slices were slightly dried, the coated tissue of secondary antibody labeled with HRP of the corresponding species of primary antibody was added to the ring and incubated at room temperature for 50 min.

7. Add iF488-TSA: The slide was placed in PBS (G2156) and washed by shaking on the pendulum table (SYC-Z100) for 3 times, 5 min each time. After the slices were slightly dried, the prepared iF488-TSA (G1231) working liquid was added to the ring and incubated for 10 min at room temperature away from light. After incubation, the slides were placed in TBST (G2150) and washed on a pendulum shaker (SYC-Z100) for 3 times, 5 min each time.

8. Antibody eluent treatment: After the sections were slightly dried, the antibody eluant restored to room temperature was spread over the whole tissue. After incubating at room temperature for 5 minutes, the antibody eluant was removed, and a sufficient amount of antibody eluant was added again to completely cover the tissue. After incubating at 37°C for 30 minutes, the slide was placed in TBST (G2150) and washed on the pendulum table (SYC-Z100) for 3 times. 5 minutes each time.

9. Serum blocking: After the section is slightly dried, 3%BSA or serum is added to the tissue and sealed at room temperature for 30 min, and the blocking reagent is determined according to the primary and secondary antibody species.

10. Add a second primary antibody: Gently shake off the sealing solution, add the primary antibody in a certain proportion to the slice, put the slice flat in a wet box (SIB-20F), incubate at 4°C overnight. (Add a small amount of water to the bottom of the wet box to prevent the antibody from evaporating)

11. Add the corresponding HRP labeled secondary antibody: The slide was placed in PBS (G2156) and washed on the pendulum shaker (SYC-Z100) for 3 times, 5 min each time. After the slices were slightly dried, the coated tissue of secondary antibody labeled with HRP of the corresponding species of primary antibody was added to the ring and incubated at room temperature for 50 min.

12. Add iF555-TSA: The slide was placed in PBS (G2156) and washed by shaking on the pendulum table (SYC-Z100) for 3 times, 5 min each time. After the slices were slightly dried, the prepared iF555-TSA (G1233) working liquid was added to the ring and incubated for 10 min at room temperature away from light. After incubation, the slides were placed in TBST (G2150) and washed on a pendulum shaker (SYC-Z100) for 3 times, 5 min each time.

13. Antibody eluent treatment: After the sections were slightly dried, the antibody eluant restored to room temperature was spread over the whole tissue. After incubating at room temperature for 5 minutes, the antibody eluant was removed, and a sufficient amount of antibody eluant was added again to completely cover the tissue. After incubating at 37°C for 30 minutes, the slide was placed in TBST (G2150) and washed on the pendulum table (SYC-Z100) for 3 times. 5 minutes each time.

Note: Steps 4-7 are one round of antibody labeling process, each round of antibody labeling only needs to change the TSA reagent with different fluorescein labeling (iF647-TSA G1232; iF594-TSA G1242; iF440-TSA G1250; iF546-TSA G1251; iF700-TSA G1252; iF750-TSA G1258). After one round of antibody labeling, incubate the antibody eluent at 37°C for 30 min, remove the primary and secondary antibodies labeled in this round, and then continue the next round of antibody labeling. Depending on the number of fluorescent multi-labeled antibodies, repeat steps 4-7 until all antibody labeling is complete.

14. DAPI restaining nuclei: After the sections were slightly dried, DAPI dye solution (G1012) was added to the circle and incubated for 10 min at room temperature away from light.

15. Autofluorescence quenching: The slide was placed in PBS (G2156) and washed by shaking on the pendulum table (SYC-Z100) for 3 times, 5 min each time. After the sections were slightly dried, the tissue self-fluorescence quencher (G1221-2) was added into the ring and incubated for 5 min in the dark, and then rinsed under water for 10 min.

16. Sealing: The section is slightly dried and sealed with anti-fluorescence quenching sealing tablet (G1401).

17. Microscopic photography: Sections are viewed under a fluorescence microscope and images are collected, or scanned with a fluorescence scanner.

 

 

1. The effect of antibody elution depends on section thickness, elution temperature and elution time. 4~8 μm slices are usually treated in a wet box at 37℃ for 30 min.

2. In order to ensure the effect of antibody elution and fluorescent multi-label labeling, it is recommended to label structural membrane protein and cytoskeleton protein first, then cytoplasmic protein, and finally nuclear protein.

3. If some antibodies have strong affinity and are not easy to elute completely (such as brain tissue GFAP), the washing temperature can be increased to 50℃ for 30 min, or the number of elution can be increased, that is, after adding antibody eluent to the tissue and incubating at 37℃ for 30 min, the antibody eluent is removed, and a new antibody eluent is added, and the incubation continues at 37℃ for 30 min.

4. This antibody eluent has strong fluidity, and when the film is not placed horizontally, the reagent is easy to flow out of the ring, affecting the elution effect. It is necessary to pay attention to the section laying flat during operation.

5. Wear gloves, mask and lab coat to avoid contact with skin and eyes. In case of contact with sensitive areas, rinse immediately with plenty of water.

6. The titer of the reagent beyond the expiry date may decrease, so please use it within the expiry date of the reagent.

 

For Research Use Only!