HSP70 Antibody Staining Protocol for Immunohistochemistry

 

 

 

Description: The HSP 70 family, Heat Shock Protein, is composed of our highly conserved proteins: HSP70, HSC70, GRP75 and GRP78. These proteins serve a variety of roles: they act as molecular chaperones facilitating the assembly of multi-protein complexes, they participate in the translocation of polypeptides across cell membranes and to the nucleus and they aid in the proper folding of nascent polypeptide chains. All members of the family, except HSP70, are constitutively expressed in primate cells. HSP70 expression is strongly induced in response to heat stress. HSP70 and HSC70 play key roles in the cytosolic endoplasmic reticulum and mitochondrial import machinery and are found in both the cytosol and nucleus of mammalian cells. They are involved in the chaperoning of nascent polypeptide chains and in protecting cells against the accumulation of improperly folded proteins. GRP78 is localized in the endoplasmic reticulum where it receives imported secretory proteins and is involved in the folding and translocation of nascent peptide chains. GRP75 expression is restricted to the mitochondrial matrix and aids in the translocation and folding of nascent polypeptide chains of both nuclear and mitochondrial origin. GRP75 and GRP78 are un-responsive to heat stress and are induced by glucose deprivation. It has been postulated that members of the HSP70 family act as force-generating motors, relying on the hydrolysis of ATP for their activity.

 

Primary Antibody

Name: HSP70 Antibody

Clone: Goat polyclonal

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-1060

Dilution: 1:200 using IHC-TekTM 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: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102)

Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100)

Heat/Cool Temperature: 95 ºC - 100 ºC/room temperature

Heat/Cool Time: 20 minutes/20 minutes

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: Cytoplasmic/nuclear

Images: Search image

Additional Information:

Tissue Type: Skin

Fixation: Formalin fixed paraffin sections

Positive Control: 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:

Not tested on frozen sections.

 

References: