Immunohistochemistry Protocol (Paraffin) for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

• Xylene
• Ethanol, anhydrous denatured, histological grade (100% and 95%)
• Deionized water (dH₂O)
• Wash Buffer:
  1. 1X Tris Buffered Saline with Tween 20 (TBST): To prepare 1 L 1X TBST, add 100 mL 10X Tris Buffered Saline with Tween 20 (TBST) #9997 to 900 mL dH₂0, mix.
  2. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS, add 100 mL 10X Wash Buffer, Phosphate Buffered Saline #12528 to 900 mL dH₂0, mix.
• SignalStain^® Antibody Diluent: #8112
• 1X EDTA Unmasking Solution: To prepare 250 mL of 1X EDTA unmasking solution, dilute 25 mL of SignalStain^® EDTA Unmasking Solution (10X) #14747 with 225 mL of dH₂O.
• Blocking Solution: 1X TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution.
  1. TBST/5% Normal Goat Serum: to 5 mL 1X TBST, add 250 µL Normal Goat Serum #5425.
  2. 1X Animal-Free Blocking Solution: to 4 mL of dH₂O, add 1 mL of Animal-Free Blocking Solution (5X) #15019.
• ProLong Gold AntiFade Reagent #9071, ProLong Gold AntiFade Reagent with DAPI #8961
• (optional) TrueBlack^® Lipofuscin Autofluorescence Quencher #92401

B. Deparaffinization/Rehydration

NOTE: Do not allow slides to dry at any time during this procedure.

1. Deparaffinize/hydrate sections:
   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.
2. Wash sections twice in dH₂O for 5 min each.

C. Antigen Unmasking

1. Heat slides, submersed in 1X EDTA unmasking solution, in a microwave until boiling begins; then follow with 15 min at sub-boiling temperature (95^°-98^°). No cooling is necessary.

D. Staining

1. Wash sections in dH₂O three times for 5 min each.
2. Incubate sections in 1X TBST for 5 min.
3. Block each section with 100-400 µL of preferred blocking solution for 1 hr at room temperature.
4. Remove blocking solution and add 100-400 µL primary antibody diluted in SignalStain^® Antibody Diluent to each section.

   NOTE: If multiplexing, combine all conjugates into a cocktail and apply in one step.

5. Incubate overnight at 4^°C.
6. Rinse three times in 1X PBS for 5 min each. Protect from light.

   NOTE: See below for optional TrueBlack^® Lipofuscin Autofluorescence Quencher protocol.

7. Coverslip slides with ProLong Gold Antifade Reagent or ProLong Gold Antifade Reagent with DAPI.
8. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4^°C. Protect from light.

TrueBlack^® Lipofuscin Autofluorescence Quencher protocol

Following Section D Step 6:

IMPORTANT: TrueBlack^® Lipofuscin Autofluorescence Quencher is not compatible with detergent. Any steps involving detergent must be done before applying TrueBlack^® Lipofuscin Autofluorescence Quencher.

1. Prepare TrueBlack^® Lipofuscin Autofluorescence Quencher solution by diluting 1:20 in 70% ethanol. Vortex to mix.

   NOTE: Quenching solution should be prepared fresh prior to use and discarded if precipitate is visible. We recommend heating the vial of stock solution of TrueBlack^® Lipofuscin Autofluorescence Quencher, 20X in DMF to 70^°C prior to dilution to prevent precipitate formation.

2. Immediately cover tissue sections with 100-200 µL of quenching solution at room temperature for 30 sec.

   IMPORTANT: Do not allow sections to dry out. Sections may be incubated for up to 3 min, provided they remain fully hydrated.

3. Tap slides on an absorbent towel to collect excess TrueBlack^® Lipofuscin Autofluorescence Quencher before transferring to 1X PBS.
4. Rinse three times in 1X PBS for 5 min each.
5. Proceed with counterstaining/mounting.

For Research Use Only. Not for Use in Diagnostic Procedures.