D. Protocol

1. Slide Baking (Optional)
   1. Incubate slides positioned horizontally for 1.5 hr at 60°C.
   2. 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.

2. Deparaffinization/Hydration
   1. Incubate sections in three washes of Xylene for 5 min each.
   2. Incubate sections in two washes of 100% ethanol for 10 min each.
   3. Incubate sections in two washes of 95% ethanol for 10 min each.
   4. Wash sections two times in dH2O for 5 min each.

   NOTE: All washes are to be done with gentle agitation at room temperature.

3. 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:

   1. Using a microwave, bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0.
   2. Maintain at a sub-boiling temperature for 10 min.
   3. Cool slides to room temperature on bench top for 30 min.

   For EDTA:

   1. Using a microwave, bring slides to a boil in 1 mM EDTA, pH 8.0.
   2. Maintain at a sub-boiling temperature for 15 min. No cooling is necessary.
4. Quenching
   1. Wash sections in dH2O three times for 5 min each.
   2. Incubate sections in 3% hydrogen peroxide for 10 min.
   3. Wash sections in dH2O two times for 5 min each.
   4. Wash sections in 1X TBST for 5 min.
5. 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.

   1. Dilute primary antibody in SignalStain® Antibody Diluent #8112.
   2. Add 100-400 μl to each section and incubate in a humidified chamber at room temperature for 60 min.
   3. During incubation with the primary antibody, equilibrate SignalStain® Boost Detection Reagent (HRP rabbit, #8114 or HRP mouse, #8125) to room temperature.
   4. Remove antibody solution and wash sections with 1X TBST two times for 3 min each.
   5. 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.
   6. Incubate in a humidified chamber at room temperature for 30 min, protected from light.
   7. Wash slides with 1X TBST two times for 3 min per wash, with gentle agitation and protected from light.
6. 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).

   1. Dilute fluorophore-conjugated TSA® Plus amplification reagent as per manufacturer’s recommendation.
   2. Apply 100-400 μl per slide and incubate for 10 min at room temperature in a humidified chamber, protected from light.
   3. Wash slides with 1X TBST two times for 3 min per wash, with gentle agitation and protected from light.
7. Serial staining
   1. If performing multiplex staining whereby more than one target of interest is to be detected:
      1. Proceed to stripping (Step 8).
   2. If you have completed your multiplex panel staining OR if you are performing a singleplex assay whereby a single target of interest is detected:
      1. Proceed to mounting (Step 9).
8. Stripping
   1. Using a microwave, bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0.
   2. Maintain at a sub-boiling temperature for 10 min.

   3. Cool slides to room temperature on bench top for 30 min.

      Proceed with Staining/Detection (Step 5) using a different tyramide-fluorophore conjugate.

9. 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.