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Fluorescent Multiplex Immunohistochemistry

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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous 40-60 Mouse IgG2a
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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human colon (ulcerative colitis) using PD-1 (EH33) Mouse mAb (IHC-Specific).

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded 293 cell pellets, control (left) or PD-1 transfected (right), using PD-1 (EH33) Mouse mAb (IHC-Specific).

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Immunohistochemistry (Paraffin)

Immunohistochemical analysis of paraffin-embedded human tonsil using PD-1 (EH33) Mouse mAb (IHC-Specific).

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Immunohistochemistry (Paraffin)

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. Hematoxylin (optional).
  5. Wash Buffer:
    1. 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
  6. SignalStain® Antibody Diluent (#8112).
  7. 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.
  8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H2O2 to 90 ml dH2O.
  9. Blocking Solution: 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).
  10. Detection System: SignalStain® Boost IHC Detection Reagents (HRP, Mouse #8125).
  11. Substrate: SignalStain® DAB Substrate Kit (#8059).
  12. Hematoxylin: Hematoxylin (#14166).
  13. Mounting Medium: SignalStain® Mounting Medium (#14177).

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

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

D. Staining

  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 wash buffer for 5 min.
  5. Block each section with 100–400 µl of preferred blocking solution for 1 hr at room temperature.
  6. Remove blocking solution and add 100–400 µl primary antibody diluted in SignalStain® Antibody Diluent (#8112) to each section. Incubate overnight at 4°C.
  7. Equilibrate SignalStain® Boost Detection Reagent (HRP, Mouse #8125) to room temperature.
  8. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
  9. Cover section with 1–3 drops SignalStain® Boost Detection Reagent (HRP, Mouse #8125) as needed. Incubate in a humidified chamber for 30 min at room temperature.
  10. Wash sections three times with wash buffer for 5 min each.
  11. Add 1 drop (30 µl) SignalStain® DAB Chromogen Concentrate to 1 ml SignalStain® DAB Diluent and mix well before use.
  12. Apply 100-400 µl SignalStain® DAB to each section and monitor closely. 1-10 minutes generally provides an acceptable staining intensity.
  13. Immerse slides in dH2O.
  14. If desired, counterstain sections with hematoxylin (#14166).
  15. Wash sections in dH2O two times for 5 min each.
  16. Dehydrate sections:
    1. Incubate sections in 95% ethanol two times for 10 sec each.
    2. Repeat in 100% ethanol, incubating sections two times for 10 sec each.
    3. Repeat in xylene, incubating sections two times for 10 sec each.
  17. Mount sections with coverslips and mounting medium (#14177).

posted February 2015

revised March 2016

Protocol Id: 664

Product Usage Information

Application Dilutions
Immunohistochemistry (Paraffin) 1:200

Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

PD-1 (EH33) Mouse mAb (IHC-Specific) recognizes transfected and endogenous levels of total PD-1 protein by immunohistochemistry on formalin-fixed paraffin-embedded tissue sections.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific to the amino terminus of human PD-1 protein.

The programmed cell death 1 protein (PD-1, PDCD1, CD279) is a member of the CD28 family of immunoreceptors that regulate T cell activation and immune responses (1-3). The PD-1 protein contains an extracellular Ig V domain, a transmembrane domain, and a cytoplasmic tail that includes an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). PD-1 is activated by the cell surface ligands PD-L1 and PD-L2 (4). Upon activation, PD-1 ITIM and ITSM phosphorylation leads to the recruitment of the protein tyrosine phosphatases SHP-1 and SHP-2, which suppress TCR signaling (5-7). In addition to activated T-cells, PD-1 is expressed in activated B-cells and monocytes, although its function in these cell types has not been fully characterized (8). The PD-1 pathway plays an important role in immune tolerance (3); however, research studies show that cancer cells often adopt this pathway to escape immune surveillance (9). Consequently, blockade of PD-1 and its ligands is proving to be a sound strategy for neoplastic intervention (10).


1.  Ishida, Y. et al. (1992) EMBO J 11, 3887-95.

2.  Shinohara, T. et al. (1994) Genomics 23, 704-6.

3.  Nishimura, H. et al. (1999) Immunity 11, 141-51.

4.  Freeman, G.J. et al. (2000) J Exp Med 192, 1027-34.

5.  Yokosuka, T. et al. (2012) J Exp Med 209, 1201-17.

6.  Sheppard, K.A. et al. (2004) FEBS Lett 574, 37-41.

7.  Chemnitz, J.M. et al. (2004) J Immunol 173, 945-54.

8.  Thibult, M.L. et al. (2013) Int Immunol 25, 129-37.

9.  Dong, H. et al. (2002) Nat Med 8, 793-800.

10.  Topalian, S.L. et al. (2012) Curr Opin Immunol 24, 207-12.


Entrez-Gene Id 5133
Swiss-Prot Acc. Q15116


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Tween is a registered trademark of ICI Americas, Inc.

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PD-1 (EH33) Mouse mAb (IHC-Specific)