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234196
Sigma-AldrichAnti-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.
More>>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. Less<<
Anti-Collagen Type X Rabbit pAb MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Recognizes 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 Number
234196
Brand Family
Calbiochem®
References
References
Chung, K.S., et al. 1995. Dev. Biol. 170, 387. Schmid. T.M., and Linsenmayer, T.M. 1985. Dev. Biol. 107, 373.
Frozen Sections (see comments) Immunoblotting (1:100-1:300) Immunoprecipitation (1:20-1:40) Paraffin Sections (see application reference; pretreatment required)
Application Comments
For 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
Immunogen
purified, rat chondrosarcoma cell collagen type X
Immunogen
Rat
Host
Rabbit
Isotype
IgG
Species Reactivity
Human
Mouse
Rat
Antibody Type
Polyclonal Antibody
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).
Anti-Collagen Type X Rabbit pAb Certificates of Analysis
Title
Lot 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.
Revision
07-August-2009 JSW
Application
Frozen Sections (see comments) Immunoblotting (1:100-1:300) Immunoprecipitation (1:20-1:40) Paraffin Sections (see application reference; pretreatment required)
Description
Rabbit polyclonal antibody supplied as undiluted serum. Recognizes type X collagen.
Background
Collagens 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.
Host
Rabbit
Immunogen species
Rat
Immunogen
purified, rat chondrosarcoma cell collagen type X
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. 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 Instructions
Following initial thaw, aliquot and freeze (-70°C).
Toxicity
Standard Handling
References
Chung, K.S., et al. 1995. Dev. Biol. 170, 387. Schmid. T.M., and Linsenmayer, T.M. 1985. Dev. Biol. 107, 373.
Application references
Paraffin Sections
Nöth, U., et al. 2007. J. Biomed. Mater. Res.83A, 626.