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73812
Hydroxyproline Antibody
Primary Antibodies
Polyclonal Antibody

Hydroxyproline Antibody #73812

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  1. IHC
Immunohistochemistry Image 1: Hydroxyproline Antibody
Immunohistochemical analysis of paraffin-embedded A-10 cell pellets, untreated (left) or treated with dehydroxyascorbic acid (right), using Hydroxyproline Antibody (top) or COL1A1 Rabbit mAb (bottom).
Immunohistochemistry Image 2: Hydroxyproline Antibody
Immunohistochemical analysis of paraffin-embedded normal human kidney (left), prostate (middle), and lung (right) using Hydroxyproline Antibody (top) or COL1A1 Rabbit mAb (bottom).
Immunohistochemistry Image 3: Hydroxyproline Antibody
Immunohistochemical analysis of paraffin-embedded normal mouse liver (left), skin (middle), and tendon (right) using Hydroxyproline Antibody (top) or COL1A1 Rabbit mAb (bottom).
Immunohistochemistry Image 4: Hydroxyproline Antibody
Immunohistochemical analysis of paraffin-embedded normal mouse liver (left) or diet-induced NASH mouse liver (right) using Hydroxyproline Antibody.
Immunohistochemistry Image 5: Hydroxyproline Antibody
Immunohistochemical analysis of paraffin-embedded human fibrosis of the stomach using Hydroxyproline Antibody (left) or COL1A1 Rabbit mAb (right).
Immunohistochemistry Image 6: Hydroxyproline Antibody
Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma (left), esophageal adenocarcinoma (middle), and prostate carcinoma (right) using Hydroxyproline Antibody (top) or COL1A1 Rabbit mAb (bottom).
Immunohistochemistry Image 7: Hydroxyproline Antibody
Dual immunohistochemical analysis of paraffin-embedded human prostate carcinoma (2 regions of interest) using E-Cadherin (4A2) Mouse mAb #14472 (brown) and Hydroxyproline Antibody (red).
To Purchase # 73812S
Product # Size Price
73812S
100 µl $ 285

Supporting Data

REACTIVITY All
SENSITIVITY Endogenous
MW (kDa)
SOURCE Rabbit

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

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
  • All-All Species Expected

Product Description

Hydroxyproline Antibody provides a means of visualizing hydroxyproline in paraffin-embedded tissues. Using Hydroxyproline Antibody in the IHC assay, global collagen in tissue can be detected, while also providing contextual information that is lost in traditional hydroxyproline assays. Additionally, Hydroxyproline Antibody can be combined with other antibodies to enable detection of other markers of interest.

Product Usage Information

Application Dilution
Immunohistochemistry (Paraffin) 1:50 - 1:200

Storage

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

Protocol

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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 dH2O, mix.
  6. SignalStain® Antibody Diluent (#8112).
  7. 1X Citrate Unmasking Solution: To prepare 250 mL of 1X citrate unmasking solution, dilute 25 ml of SignalStain® Citrate Unmasking Solution (10X) (#14746) 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, Rabbit #8114).
  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 Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

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, Rabbit #8114) 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, Rabbit #8114) 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. 5–10 min 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, Rabbit) #8114 SignalStain® Boost IHC Detection Reagent (AP, Rabbit) #18653
COMPATIBLE
CHROMOGEN
SignalStain® DAB Substrate Kit #8059 SignalStain® Vibrant Red Alkaline Phosphatase Substrate Kit #76713
SignalStain® Vivid Purple Peroxidase Substrate Kit #96632  

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 2010

revised April 2020

Protocol Id: 1989

Specificity / Sensitivity

Hydroxyproline Antibody recognizes endogenous levels of proline only when the proline is hydroxylated. The antibody does not react with non-hydroxylated proline.

Species Reactivity:

All Species Expected

Source / Purification

Polyclonal antibodies are produced by immunizing animals with an antigen containing hydroxyproline. Antibodies are purified by affinity chromatography.

Background

Collagens are a large family of proteins and collectively they are the most abundant protein in mammals. They are trimeric molecules comprised of three alpha polypeptide chains that form a triple helix structure that is characteristic of all collagens (1). Collagen chains have important features that allow helix to form: every third amino acid in the sequence is a glycine, and the chains are co- and post-translationally modified. Prolines are hydroxylated by collagen prolyl 4-hydroxylases. These hydroxyproline modifications are critical for the stability of the triple helix to form fibrils (2). While proline hydroxylation occurs on a small number of other proteins, the hydroxyproline content of tissue comes almost entirely from collagen, such that hydroxyproline quantitative biochemical assays are routinely utilized as a measurement of collagen (3).
  1. Gordon, M.K. and Hahn, R.A. (2010) Cell Tissue Res 339, 247-57.
  2. Rappu, P. et al. (2019) Essays Biochem 63, 325-35.
  3. Cissell, D.D. et al. (2017) Tissue Eng Part C Methods 23, 243-50.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

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's 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's 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.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SignalStain is a trademark of Cell Signaling Technology, Inc.