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234196 Anti-Collagen Type X Rabbit pAb

This Anti-Collagen Type X Rabbit pAb is validated for use in Frozen Sections, Immunoblotting, Immunoprecipitation, Paraffin Sections for the detection of Collagen Type X.

Anti-Collagen Type X Rabbit pAb MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
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234196
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Overview

Replacement Information

Key Spec Table

Species ReactivityHostAntibody Type
H, M, RRbPolyclonal Antibody

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      Description
      OverviewRecognizes type X collagen. Exhibits slight cross-reactivity with fibronectin and type II and type IX collagen. Does not cross-react with type I, type III, or type XI collagen.
      Catalogue Number234196
      Brand Family Calbiochem®
      References
      ReferencesChung, K.S., et al. 1995. Dev. Biol. 170, 387.
      Schmid. T.M., and Linsenmayer, T.M. 1985. Dev. Biol. 107, 373.
      Product Information
      FormLiquid
      FormulationUndiluted serum.
      PreservativeNone
      Quality LevelMQ100
      Applications
      Application ReferencesParaffin Sections Nöth, U., et al. 2007. J. Biomed. Mater. Res. 83A, 626.
      Key Applications Frozen Sections
      Immunoblotting (Western Blotting)
      Immunoprecipitation
      Paraffin Sections
      Application NotesFrozen Sections (see comments)
      Immunoblotting (1:100-1:300)
      Immunoprecipitation (1:20-1:40)
      Paraffin Sections (see application reference; pretreatment required)
      Application CommentsFor frozen sections, enzymatic or heat (41°C) pretreatment is recommended to unwind the triple helical structure. The optimal dilution for frozen sections will vary with sample type. For staining paraffin sections, pretreatment with 0.25% trypsin containing 1 mM EDTA, 37°C is recommended (see application reference). Exhibits slight cross-reactivity to fibronectin and collagen types II and IX. Does not cross-react with collagen types I, III, or XI. Variables associated with assay conditions will dictate the proper working dilution.

      This protocol is provided only as a general guide. Researchers should standardize this assay for their own specific needs and should consult published literature.

      Immunohistochemistry Protocol

      Introduction

      Staining of cryosections using anti-collagen antibodies requires either heat and/or enzymatic treatment in order to unwind the triple helical structure of collagen. An initial heat treatment aids in unwinding the collagen, while maintaining the antigen structure. Subsequent enzymatic treatment with hyaluronidase facilitates the removal of macromolecules that further hinder recognition by the antibody. In some cases it is also necessary to perform a second enzymatic digestion using chondroitinase ABC.

      Protocol

      Reagents and Equipment:

      • Cryostat sections of desired tissue: cut into 5 mm sections and place on clean glass slides treated with poly-L-lysine
      • PBS (phosphate buffered saline): dissolve 8 g NaCl, 200 mg KCl, 1.44 g Na2HPO4, and 240 mg KH2PO4 in 800 ml dH2O; adjust the pH to 7.4 with HCl; add dH2O to 1 L
      • PBS/BSA (phosphate buffered saline/bovine serum albumin): 0.5% BSA prepared in PBS
      • Testicular hyaluronidase: 2 mg/ml in cold PBS
      • Chondroitinase ABC: 2 mg/ml in PBS
      • Normal sheep serum
      • Primary antibody: Anti-Collagen, Type X Rabbit pAb (Cat. No. 234196); diluted as recommended
      • Secondary antibody: goat anti-rabbit IgG, fluorescein conjugated
      • p-Phenylenediamine: 0.1% in glycerin:PBS (9:1)
      • Staining chamber
      • Wet chamber for incubations: chamber in which a small amount of PBS or dH2O is added to maintain a humid environment; slides should not be submerged

      Protocol:

      • Prepare 5 µm cryostat tissue sections from the tissue of choice and place on clean glass slides that have been treated with poly-L-lysine.
      • Incubate the tissue sections in a staining chamber with 0.5% BSA/PBS for 5-10 min at 41°-43°C. This serves to block the sections and unwind the helical structure.
      • Carefully rinse by briefly submerging in PBS.
      • Wash 2 x 10 min in PBS. Carefully remove excess fluid and air-dry the slides.
      • Treat the sections with 10 µl of hyaluronidase stock (2 mg/ml) by applying the enzyme directly to the sections on the slide. If 10 µl does not completely cover the section, increase the volume of enzyme until the section is completely covered. Incubate in a wet chamber at room temperature for 30 min. Stop the reaction by treatment of the sections with 25-50 µl of normal sheep serum.
      • Carefully rinse by briefly submerging in PBS.
      • Wash 2 x 10 min in PBS. Carefully remove excess fluid and air-dry the slides.
      • If desired, a second enzymatic treatment can be carried out by treating the sections with 10 µl of chondroitinase ABC (2 mg/ml); apply directly to the sections as above. If 10 µl does not completely cover the section, increase the volume of enzyme until the section is completely covered. Incubate in a wet chamber at room temperature for 30 min. Stop the reaction by treatment of the sections with 25-50 µl of normal sheep serum.
      • Carefully rinse by briefly submerging in PBS.
      • Wash 2 x 10 min in PBS. Carefully remove excess fluid and air-dry the slides.
      • Apply 5-10 µl of primary antibody (diluted to the desired concentration) to the section and incubate in a wet chamber at 37°C for 45 min.
      • Carefully rinse by briefly submerging in PBS.
      • Wash 2 x 10 min in PBS. Carefully remove excess fluid and air-dry the slides.
      • Apply 5-10 µl of secondary antibody (diluted to the desired concentration) to the section and incubate in a wet chamber at 37°C for 1 h. Protect from light during the incubation.
      • Carefully rinse by briefly submerging in PBS.
      • Wash 2 x 10 min in PBS in a staining chamber. Carefully remove excess fluid, but do not allow the slides to completely dry, as the secondary antibody may form crystals.
      • Place 1 drop of 0.1% p-phenylenediamine on the section and mount under a coverslip. The slides can be stored at 4°C up to 2 days.
      • Fixation:
      The best results have been obtained on cryosections without fixation. The risk of collagen loss is relatively low, as its solubility in neutral buffer is negligible. However, other antigens may diffuse into the incubation medium and settle along adjacent tissue structures or form diffusion artifacts. If fixation is necessary, dehydration with ethanol or acetone at -20°C for 5-10 min is recommended. Fixation should take place following enzymatic treatment (step 5). Aldehyde (formaldehyde or glutaraldehyde) fixation is NOT recommended. If aldehyde fixation cannot be avoided, do not exceed 2% of the fixative (less than 1% is recommended).

      Notes:

      • It may not be necessary to treat the sections with both heat and hyaluronidase and/or chondroitinase ABC. It is recommended that heat treatment be used initially and, if further epitope exposure is necessary, then treat with hyaluronidase. Chondroitinase ABC treatment is usually not necessary.
      • If hyaluronidase and chondroitinase ABC are both desired, it is recommended that the digestions be carried out separately.
      Biological Information
      Immunogenpurified, rat chondrosarcoma cell collagen type X
      ImmunogenRat
      HostRabbit
      IsotypeIgG
      Species Reactivity
      • Human
      • Mouse
      • Rat
      Antibody TypePolyclonal Antibody
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Standard Handling
      Storage ≤ -70°C
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
      Packaging Information
      Transport Information
      Supplemental Information
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      234196-500ULCN 04055977200119

      Documentation

      Anti-Collagen Type X Rabbit pAb SDS

      Title

      Safety Data Sheet (SDS) 

      Anti-Collagen Type X Rabbit pAb Certificates of Analysis

      TitleLot Number
      234196

      References

      Reference overview
      Chung, K.S., et al. 1995. Dev. Biol. 170, 387.
      Schmid. T.M., and Linsenmayer, T.M. 1985. Dev. Biol. 107, 373.
      Data Sheet

      Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

      Revision07-August-2009 JSW
      ApplicationFrozen Sections (see comments)
      Immunoblotting (1:100-1:300)
      Immunoprecipitation (1:20-1:40)
      Paraffin Sections (see application reference; pretreatment required)
      DescriptionRabbit polyclonal antibody supplied as undiluted serum. Recognizes type X collagen.
      BackgroundCollagens are major fibrous structural elements of cartilage, tendon, bone, skin, lung, and blood vessels. Type X collagen, a heterotrimer consisting of three α1(X) subunits, is found in hypertrophic and mineralizing cartilage. Type X collagen provides a transition to bone by allowing removal of hypertrophic cartilage and calcification. Type X collagen is believed to be involved in skeletal growth, maintenance, and repair.
      HostRabbit
      Immunogen speciesRat
      Immunogenpurified, rat chondrosarcoma cell collagen type X
      IsotypeIgG
      Specieshuman, mouse, rat
      FormLiquid
      FormulationUndiluted serum.
      PreservativeNone
      CommentsFor frozen sections, enzymatic or heat (41°C) pretreatment is recommended to unwind the triple helical structure. The optimal dilution for frozen sections will vary with sample type. For staining paraffin sections, pretreatment with 0.25% trypsin containing 1 mM EDTA, 37°C is recommended (see application reference). Exhibits slight cross-reactivity to fibronectin and collagen types II and IX. Does not cross-react with collagen types I, III, or XI. Variables associated with assay conditions will dictate the proper working dilution.

      This protocol is provided only as a general guide. Researchers should standardize this assay for their own specific needs and should consult published literature.

      Immunohistochemistry Protocol

      Introduction

      Staining of cryosections using anti-collagen antibodies requires either heat and/or enzymatic treatment in order to unwind the triple helical structure of collagen. An initial heat treatment aids in unwinding the collagen, while maintaining the antigen structure. Subsequent enzymatic treatment with hyaluronidase facilitates the removal of macromolecules that further hinder recognition by the antibody. In some cases it is also necessary to perform a second enzymatic digestion using chondroitinase ABC.

      Protocol

      Reagents and Equipment:

      • Cryostat sections of desired tissue: cut into 5 mm sections and place on clean glass slides treated with poly-L-lysine
      • PBS (phosphate buffered saline): dissolve 8 g NaCl, 200 mg KCl, 1.44 g Na2HPO4, and 240 mg KH2PO4 in 800 ml dH2O; adjust the pH to 7.4 with HCl; add dH2O to 1 L
      • PBS/BSA (phosphate buffered saline/bovine serum albumin): 0.5% BSA prepared in PBS
      • Testicular hyaluronidase: 2 mg/ml in cold PBS
      • Chondroitinase ABC: 2 mg/ml in PBS
      • Normal sheep serum
      • Primary antibody: Anti-Collagen, Type X Rabbit pAb (Cat. No. 234196); diluted as recommended
      • Secondary antibody: goat anti-rabbit IgG, fluorescein conjugated
      • p-Phenylenediamine: 0.1% in glycerin:PBS (9:1)
      • Staining chamber
      • Wet chamber for incubations: chamber in which a small amount of PBS or dH2O is added to maintain a humid environment; slides should not be submerged

      Protocol:

      • Prepare 5 µm cryostat tissue sections from the tissue of choice and place on clean glass slides that have been treated with poly-L-lysine.
      • Incubate the tissue sections in a staining chamber with 0.5% BSA/PBS for 5-10 min at 41°-43°C. This serves to block the sections and unwind the helical structure.
      • Carefully rinse by briefly submerging in PBS.
      • Wash 2 x 10 min in PBS. Carefully remove excess fluid and air-dry the slides.
      • Treat the sections with 10 µl of hyaluronidase stock (2 mg/ml) by applying the enzyme directly to the sections on the slide. If 10 µl does not completely cover the section, increase the volume of enzyme until the section is completely covered. Incubate in a wet chamber at room temperature for 30 min. Stop the reaction by treatment of the sections with 25-50 µl of normal sheep serum.
      • Carefully rinse by briefly submerging in PBS.
      • Wash 2 x 10 min in PBS. Carefully remove excess fluid and air-dry the slides.
      • If desired, a second enzymatic treatment can be carried out by treating the sections with 10 µl of chondroitinase ABC (2 mg/ml); apply directly to the sections as above. If 10 µl does not completely cover the section, increase the volume of enzyme until the section is completely covered. Incubate in a wet chamber at room temperature for 30 min. Stop the reaction by treatment of the sections with 25-50 µl of normal sheep serum.
      • Carefully rinse by briefly submerging in PBS.
      • Wash 2 x 10 min in PBS. Carefully remove excess fluid and air-dry the slides.
      • Apply 5-10 µl of primary antibody (diluted to the desired concentration) to the section and incubate in a wet chamber at 37°C for 45 min.
      • Carefully rinse by briefly submerging in PBS.
      • Wash 2 x 10 min in PBS. Carefully remove excess fluid and air-dry the slides.
      • Apply 5-10 µl of secondary antibody (diluted to the desired concentration) to the section and incubate in a wet chamber at 37°C for 1 h. Protect from light during the incubation.
      • Carefully rinse by briefly submerging in PBS.
      • Wash 2 x 10 min in PBS in a staining chamber. Carefully remove excess fluid, but do not allow the slides to completely dry, as the secondary antibody may form crystals.
      • Place 1 drop of 0.1% p-phenylenediamine on the section and mount under a coverslip. The slides can be stored at 4°C up to 2 days.
      • Fixation:
      The best results have been obtained on cryosections without fixation. The risk of collagen loss is relatively low, as its solubility in neutral buffer is negligible. However, other antigens may diffuse into the incubation medium and settle along adjacent tissue structures or form diffusion artifacts. If fixation is necessary, dehydration with ethanol or acetone at -20°C for 5-10 min is recommended. Fixation should take place following enzymatic treatment (step 5). Aldehyde (formaldehyde or glutaraldehyde) fixation is NOT recommended. If aldehyde fixation cannot be avoided, do not exceed 2% of the fixative (less than 1% is recommended).

      Notes:

      • It may not be necessary to treat the sections with both heat and hyaluronidase and/or chondroitinase ABC. It is recommended that heat treatment be used initially and, if further epitope exposure is necessary, then treat with hyaluronidase. Chondroitinase ABC treatment is usually not necessary.
      • If hyaluronidase and chondroitinase ABC are both desired, it is recommended that the digestions be carried out separately.
      Storage Avoid freeze/thaw
      ≤ -70°C
      Do Not Freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
      Toxicity Standard Handling
      ReferencesChung, K.S., et al. 1995. Dev. Biol. 170, 387.
      Schmid. T.M., and Linsenmayer, T.M. 1985. Dev. Biol. 107, 373.
      Application referencesParaffin Sections Nöth, U., et al. 2007. J. Biomed. Mater. Res. 83A, 626.