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234167 Sigma-AldrichAnti-Collagen Type I Rabbit pAb

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

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

SDS
CoA
References
Data Sheet

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234167

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Overview

Replacement Information

Key Spec Table

Species Reactivity	Host	Antibody Type
H, M, R	Rb	Polyclonal Antibody

Description
Overview	Recognizes type I collagen. Exhibits slight cross-reactivity with type III collagen.
Catalogue Number	234167
Brand Family	Calbiochem®

References
References	Romanos, G.E., et al. 1992. J. Periodont. Res. 27, 101. Zimmermann, B., et al. 1992. Eur. Arch. Biol. 103, 93. Schröter-Kermani, C., et al. 1991. Matrix 11, 428.

Product Information
Form	Liquid
Formulation	Undiluted serum.
Preservative	None
Quality Level	MQ100

Applications
Key Applications	Frozen Sections Immunoblotting (Western Blotting) Immunofluorescence Immunoprecipitation
Application Notes	Frozen Sections (1:30-1:60, see comments) Immunoblotting (1:200-1:500) Immunofluorescence (1:20-1:40) Immunoprecipitation (1:20-1:60)
Application Comments	For frozen sections, enzymatic or heat (41°C) pretreatment is recommended to unwind the triple helical structure. Exhibits very slight cross-reactivity with collagen type III. 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 I Rabbit pAb (Cat. No. 234167); 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
Immunogen	purified, fetal mouse skin collagen type I
Immunogen	Fetal Mouse Skin
Host	Rabbit
Isotype	IgG
Species Reactivity	Human Mouse Rat
Antibody Type	Polyclonal 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 Instructions	Following initial thaw, aliquot and freeze (-70°C).

Packaging Information

Transport Information

Supplemental Information

Specifications

Global Trade Item Number
Catalogue Number	GTIN
234167	0

Documentation

Anti-Collagen Type I Rabbit pAb SDS

Title
Safety Data Sheet (SDS)

Anti-Collagen Type I Rabbit pAb Certificates of Analysis

Title	Lot Number
234167	Enter Lot Number

References

Reference overview
Romanos, G.E., et al. 1992. J. Periodont. Res. 27, 101. Zimmermann, B., et al. 1992. Eur. Arch. Biol. 103, 93. Schröter-Kermani, C., et al. 1991. Matrix 11, 428.

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.

Revision	26-July-2007 RFH
Application	Frozen Sections (1:30-1:60, see comments) Immunoblotting (1:200-1:500) Immunofluorescence (1:20-1:40) Immunoprecipitation (1:20-1:60)
Description	Rabbit polyclonal antibody supplied as undiluted serum. Recognizes the ~115 kDa (doublet) type I collagen protein.
Background	Collagens are major fibrous structural elements of cartilage, tendon, bone, skin, lung, and blood vessels. Type I collagen, a heterotrimer consisting of two α1 chains and one α2 chain, is the most abundant fibrillar collagen. It is preferentially synthesized in bone, skin, and tendon and is the most abundant protein found in these tissues.
Host	Rabbit
Immunogen species	Fetal Mouse Skin
Immunogen	purified, fetal mouse skin collagen type I
Isotype	IgG
Species	human, mouse, rat
Form	Liquid
Formulation	Undiluted serum.
Preservative	None
Comments	For frozen sections, enzymatic or heat (41°C) pretreatment is recommended to unwind the triple helical structure. Exhibits very slight cross-reactivity with collagen type III. 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 I Rabbit pAb (Cat. No. 234167); 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 Instructions	Following initial thaw, aliquot and freeze (-70°C).
Toxicity	Standard Handling
References	Romanos, G.E., et al. 1992. J. Periodont. Res. 27, 101. Zimmermann, B., et al. 1992. Eur. Arch. Biol. 103, 93. Schröter-Kermani, C., et al. 1991. Matrix 11, 428.

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