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43248
PD-1 (EH33) Mouse mAb
一抗
单克隆抗体

PD-1 (EH33) Mouse mAb #43248

Citations (25)
Filter:
  1. IHC
Immunohistochemical analysis of paraffin-embedded human colon adenocarcinoma using PD-1 (EH33) Mouse mAb performed on the Leica® BOND Rx.
Immunohistochemical analysis of paraffin-embedded human colon (ulcerative colitis) using PD-1 (EH33) Mouse mAb.
Immunohistochemical analysis of paraffin-embedded human tonsil using PD-1 (EH33) Mouse mAb.
Immunohistochemical analysis of paraffin-embedded 293 cell pellets, control (left) or PD-1 transfected (right), using PD-1 (EH33) Mouse mAb.
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).
To Purchase # 43248S
Cat. # Size Price Inventory
43248T
20 µl
43248S
100 µl

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Mouse IgG2a

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • C&R-CUT&RUN
  • C&T-CUT&Tag
  • DB-Dot Blot
  • eCLIP-eCLIP
  • IF-Immunofluorescence
  • F-Flow Cytometry

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • GP-Guinea Pig
  • Rab-Rabbit
  • All-All Species Expected

Product Usage Information

Application Dilution
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.

For a carrier-free (BSA and Azide) version of this product see product #60847.

Protocol

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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).

DETECTION REAGENT/SUBSTRATE COMPATIBILITY
RECOMMENDED
DETECTION REAGENTS
SignalStain® Boost IHC Detection Reagent (HRP, Mouse) #8125 SignalStain® Boost IHC Detection Reagent (AP, Mouse) #31926
COMPATIBLE
CHROMOGEN
SignalStain® DAB Substrate Kit #8059 SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713
SignalStain® Vivid Purple Peroxidase Substrate Kit #96632 SignalStain® Ultra Blue Alkaline Phosphatase Substrate Kit #12824
SignalStain® Deep Black Peroxidase Substrate Kit #72986  
SignalStain® Radiant Yellow Peroxidase Substrate Kit #69644  

NOTE: Use of detection reagents other than those specified in this protocol may require further optimization of the primary antibody to account for the different sensitivities of the detection reagents.


posted February 2015

revised June 2020

Protocol Id: 664

Specificity / Sensitivity

PD-1 (EH33) Mouse mAb 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.

Background

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).

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

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