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).
Immunohistochemical analysis of paraffin-embedded normal human kidney (left), prostate (middle), and lung (right) using Hydroxyproline Antibody (top) or COL1A1 Rabbit mAb (bottom).
Immunohistochemical analysis of paraffin-embedded normal mouse liver (left), skin (middle), and tendon (right) using Hydroxyproline Antibody (top) or COL1A1 Rabbit mAb (bottom).
Immunohistochemical analysis of paraffin-embedded normal mouse liver (left) or diet-induced NASH mouse liver (right) using Hydroxyproline Antibody.
Immunohistochemical analysis of paraffin-embedded human fibrosis of the stomach using Hydroxyproline Antibody (left) or COL1A1 Rabbit mAb (right).
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).
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).
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.
|Immunohistochemistry (Paraffin)||1:50 - 1:200|
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.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
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.
|SignalStain® Boost IHC Detection Reagent (HRP, Rabbit) #8114||SignalStain® Boost IHC Detection Reagent (AP, Rabbit) #18653|
|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
Hydroxyproline Antibody recognizes endogenous levels of proline only when the proline is hydroxylated. The antibody does not react with non-hydroxylated proline.
All Species Expected
Polyclonal antibodies are produced by immunizing animals with an antigen containing hydroxyproline. Antibodies are purified by affinity chromatography.
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).
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