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43248
PD-1 (EH33) Mouse mAb (IHC-Specific)
Primary Antibodies

PD-1 (EH33) Mouse mAb (IHC-Specific) #43248

APPLICATIONS

REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Mouse IgG2a
IHC-Leica® Bond™

Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using PD-1 (EH33) Mouse mAb (IHC-Specific) performed on the Leica® BOND™ Rx.

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IHC-P (paraffin)

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

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IHC-P (paraffin)

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

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IHC-P (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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IHC-P (paraffin)

Multiplex immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using PD-1 (EH33) mouse mAb (green), CD8α (C8/144B) mouse mAb #70306 (magenta), CD68 (D4B9C) XP® rabbit mAb #76437 (red), Pan-keratin (C11) mouse mAb #4545 (cyan), LAG3 (D2G4O™) XP® rabbit mAb #15372 (orange), and TIM-3 (D5D5R™) XP® rabbit mAb #45208 (yellow).

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Immunohistochemistry (Leica®BOND™)

NOTE: Please see product datasheet or product webpage for appropriate antibody dilution^.

  Step Reagents Time/Temperature
1 Dewax BOND™ Dewax Solution, 100% Alcohol, BOND™ Wash Solution Pre-programmed Leica® BOND™ 
2 Antigen Retrieval  BOND™ Epitope Retrieval ER2 Solution 20 min., 100˚C | Protocol: HIER 20 min with ER2
3 Peroxide Block Refine Detection Kit Peroxide Block* 5 min.
  WASH BOND™ Wash Solution  3x 0:00 min.
4 Protein Block (optional) #5425 NGS or #15019 Animal-Free Blocking Solution   20 min. 
5 Primary Antibody^ Dilute in #8112 SignalStain® Antibody Diluent 60 min. 
  WASH BOND™ Wash Solution  3x 2:00 min.
6 Post Primary Mouse Linker Refine Detection Kit Post Primary*  10 min. 
  WASH BOND™ Wash Solution  3x 2:00 min.
7 Secondary Detection Refine Detection Kit Polymer*  10 min. 
  WASH BOND™ Wash Solution/Deionized Water Custom (see below)
8a Visualization Refine Detection Kit Mixed DAB Refine*  0:00 min. 
8b Visualization Refine Detection Kit Mixed DAB Refine*  10 min. 
  WASH Deionized Water 3x 0:00 min.
9 Counterstain Refine Detection Kit Hematoxylin*  5 min. 
  WASH Deionized Water 0:00 min. 
  WASH BOND™ Wash Solution  0:00 min. 
  WASH Deionized Water 0:00 min. 
10 Dehydration (Offline):    
  Incubate sections in 95% ethanol two times for 10 seconds each.  
  Repeat in 100% ethanol, incubating sections two times for 10 seconds each.  
  Repeat in xylene, incubating sections two times for 10 seconds each.  
11 Mount sections with coverslips and #14177 SignalStain® Mounting Medium  
       
  Optional Custom wash:  BOND™ Wash Solution 2:00
    BOND™ Wash Solution Dispenser Type: OPEN 0:00
    BOND™ Wash Solution 2:00
    BOND™ Wash Solution Dispenser Type: OPEN 0:00
    BOND™ Wash Solution 0:00
    Deionized Water 0:00

*Reagent included in BOND™ Polymer Refine Detection Kit (Catalog No: DS9800)

LEICA® is a registered trademark of Leica Microsystems IR GmbH.

BOND™ is a trademark of Leica Biosystems Melbourne Pty. Ltd. No affiliation or sponsorship between CST and Leica Microsystems IR GmbH or Leica Biosystems Melbourne Pty. Ltd is implied.

posted August 2018

revised September 2018

Protocol Id: 1484

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

Application Dilutions
IHC-Leica® Bond™ 1:100
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.

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

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.
BOND is a trademark of Leica Biosystems Melbourne Pty. Ltd. No affiliation or sponsorship between CST and Leica Microsystems IR GmbH or Leica Biosystems Melbourne Pty. Ltd is implied.
LEICA is a registered trade​mark of Leica Microsystems IR GmbH.
Tween is a registered trademark of ICI Americas, Inc.

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