Collagen Type IV Antibody Staining Protocol for Immunohistochemistry

Description: Collagen type IV is a major component of basement membranes, such as those associated with capillaries and within glomeruli. This antibody will help detect collagen type IV and determine the presence of an intact basal lamina in normal tissues or the loss of structure as seen in various carcinomas.

Primary Antibody

Name: Collagen Type IV Antibody

Clone: PHM-12, Mouse anti-Human

Supplier: Vector Labs

Catalog Number: VP-C388

Dilution: 1:100 using IHC-Tek^TM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60 minutes/room temperature

Antigen Retrieval

Device: Steamer, Incubator

Buffer/pH value: Citrate buffer/pH 6.0 for 20 minutes and let sections cool for 5-10 minutes. Then followed immediately by Proteinase K digestion for 30 seconds at 37 °C. Cool sections for another 10 minutes at room temperature.

Heat/Cool Temperature: 95-100 ºC/room temperature for citrate buffer retrieval, 37ºC/room temperature for proteinase K digestion.

Heat/Cool Time: follow instruction above

Detection Methods

Standard Method: ABC Method or LSAB Method

Enhanced Method: Polymeric Methods

Chromogen Substrate

Reagent: DAB

Incubation Time/Temperature: 1-3 minutes/room temperature

Counterstain

Reagent: Mayer's Hematoxylin

Staining Time: 30 seconds

Results:

Staining Pattern: Membrane

Images: Search image

Additional Information:

Tissue Type: Kidney, skin

Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections

Positive Control: Kidney, skin

Negative Control: Omit primary antibody, isotype control or absorption control

Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary

Notes:

There is no need to perform antigen retrieval on frozen sections

References: