Immunohistochemistry Protocol (Paraffin) for Chimeric Antibodies

A. Solutions and Reagents

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

1. Xylene
2. Ethanol, anhydrous denatured, histological grade (100% and 95%)
3. Deionized water (dH₂O)
4. 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 dH₂O, mix.
2. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS, add 100 mL 10X Wash Buffer, Phosphate Buffered Saline (PBS) #12528 to 900 mL dH₂O, mix.
5. SignalStain^® Antibody Diluent: #8112
6. 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.
7. Blocking Solution: 1X TBST/5% Normal Goat Serum or 1X Animal-Free Blocking Solution
   1. 1X TBST/5% Normal Goat Serum: add 250 µL Normal Goat Serum #5425 to 5 mL 1X TBST.
   2. 1X Animal-Free Blocking Solution: add 1 mL of Animal-Free Blocking Solution (5X) #15019 to 4 mL dH₂O.
8. Fluorochrome-conjugated Secondary Antibody

IMPORTANT: Recombinant chimeric monoclonal antibodies retain the antigen-binding Fab region of the original parent host sequence from which they are engineered. When multiplexing, rabbit Fc-directed secondaries are required to detect rabbit-host primary antibodies. For more information, please refer to the Source/Purification section on the product webpage or datasheet.

When using rabbit Fc-directed secondaries, a higher concentration of primary antibody may be required. The optimal dilution of each primary antibody should be determined by titration.

Additionally, it is critical to understand the species reactivity profiles of the secondary antibodies being used in multiplexing experiments. If not using secondary antibodies suggested by Cell Signaling Technology, a thorough characterization of the secondary antibodies is recommended before combining primary antibodies with different host species.

9. Mounting Medium: Use ProLong Gold AntiFade Reagent #9071, Prolong Gold AntiFade Reagent with DAPI #8961, or DAPI #4083.

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 two times in dH₂O for 5 min each.

C. Antigen Unmasking

1. Heat slides submerged in 1X EDTA unmasking solution in a microwave until boiling begins; 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. Wash 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. To each section, add 100-400 µL of primary antibody (or antibody cocktail if multiplexing) diluted in SignalStain^® Antibody Diluent #8112.
5. Incubate overnight at 4°C.
6. Rinse three times in 1X PBS for 5 min each, protected from light.
7. Incubate samples with fluorochrome-conjugated secondary antibody (or secondary antibody cocktail if multiplexing) diluted in SignalStain^® Antibody Diluent #8112 for 1 hr at room temperature in the dark.
8. Rinse three times in 1X PBS for 5 min each, protected from light.
9. If mounting with ProLong Gold Antifade Reagent with DAPI #8961, omit this step: Prepare DAPI solution per manufacturer's recommendation and add to each section, then incubate for 5 min protected from light.
10. Rinse three times in 1X PBS for 5 min each, protected from light.
11. Coverslip slides with ProLong Gold Antifade Reagent.
12. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

_{For Research Use Only. Not for Use in Diagnostic Procedures.}