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님의 질문을 정리해볼께요:
You want to do double-IHC and have two primary antibodies (AB). The host of 1st primary Ab is rabbit. The host of 2nd primary Ab is rabbit. Can I use both for double IHC?
맞아요?
대답: 아니오. Find out one of the two antibodies whose host is either mouse or rat.
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qwerty
(비회원)
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18.05.11 07:42
D. Protocol
- Slide Baking (Optional)
- Incubate slides positioned horizontally for 1.5 hr at 60°C.
- Re-position the slides from a horizontal to an upright position and incubate for an additional 30 min at 60°C.
NOTE: This step allows for the paraffin wax to melt.
- Deparaffinization/Hydration
- Incubate sections in three washes of Xylene for 5 min each.
- Incubate sections in two washes of 100% ethanol for 10 min each.
- Incubate sections in two washes of 95% ethanol for 10 min each.
- Wash sections two times in dH2O for 5 min each.
NOTE: All washes are to be done with gentle agitation at room temperature.
- Antigen Unmasking
NOTE: Consult product data sheet for a recommendation on the optimal unmasking solution to use for each primary antibody in a multiplex panel. If the multiplex panel includes one antibody that is recommended for use with EDTA retrieval, use EDTA as the unmasking solution.
For Citrate:
- Using a microwave, bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0.
- Maintain at a sub-boiling temperature for 10 min.
- Cool slides to room temperature on bench top for 30 min.
For EDTA:
- Using a microwave, bring slides to a boil in 1 mM EDTA, pH 8.0.
- Maintain at a sub-boiling temperature for 15 min. No cooling is necessary.
- Quenching
- Wash sections in dH2O three times for 5 min each.
- Incubate sections in 3% hydrogen peroxide for 10 min.
- Wash sections in dH2O two times for 5 min each.
- Wash sections in 1X TBST for 5 min.
- Staining/Detection
NOTE: A separate pre-blocking of tissue sections may be performed but is not necessary. Optimal dilutions of the primary antibody must be determined empirically.
- Dilute primary antibody in SignalStain® Antibody Diluent #8112.
- Add 100-400 μl to each section and incubate in a humidified chamber at room temperature for 60 min.
- During incubation with the primary antibody, equilibrate SignalStain® Boost Detection Reagent (HRP rabbit, #8114 or HRP mouse, #8125) to room temperature.
- Remove antibody solution and wash sections with 1X TBST two times for 3 min each.
- Cover tissue sections in several drops (100-400 μl) of SignalStain® Boost IHC detection Reagent (HRP rabbit, #8114 or HRP mouse, #8125) specific to the species of the primary antibody.
- Incubate in a humidified chamber at room temperature for 30 min, protected from light.
- Wash slides with 1X TBST two times for 3 min per wash, with gentle agitation and protected from light.
- Tyramide Signal Amplification (TSA®)
NOTE: When choosing the appropriate fluorophore-conjugated TSA® Plus amplification reagent, it is important to consider target expression levels and fluorophore intensity. Optimal pairing of primary antibody and fluorophore should be established in advance (see Important tips above).
- Dilute fluorophore-conjugated TSA® Plus amplification reagent as per manufacturer’s recommendation.
- Apply 100-400 μl per slide and incubate for 10 min at room temperature in a humidified chamber, protected from light.
- Wash slides with 1X TBST two times for 3 min per wash, with gentle agitation and protected from light.
- Serial staining
- If performing multiplex staining whereby more than one target of interest is to be detected:
- Proceed to stripping (Step 8).
- If you have completed your multiplex panel staining OR if you are performing a singleplex assay whereby a single target of interest is detected:
- Proceed to mounting (Step 9).
- Stripping
- Using a microwave, bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0.
- Maintain at a sub-boiling temperature for 10 min.
Cool slides to room temperature on bench top for 30 min.
Proceed with Staining/Detection (Step 5) using a different tyramide-fluorophore conjugate.
- Mounting
Mount sections with coverslips using ProLong® Gold Antifade Reagent with DAPI #8961.
NOTE: If slides are being used for the purpose of constructing a spectral library, ProLong® Gold Antifade Reagent #9071 should be used.
double staining을 위해서는 1차 항체를 host가 다른것으로 바꾸셔야 할것 같습니다.
같으면 안되니까 안티바디를 새로 사셔야 합니다.
호스트가 같으면 당연히 2차 붙일때 구분없이 다 붙어버리니까요.