Citrate buffer（ph=6.0）. this works really good in our lab.

Thank you for your reply Dayan. I routinely use citrate buffer for most antibodies but This does not work for staining the chicken endothelial cells.

I have tried trypsin and pronase digestion instead but not so good result.

Hi Saritha,

collegues of mine used sambucus nigra lectin to label blood vessels in the chick embryo. Hope that helps.

Besides a lower pH buffer like citrate, we routinely use  higher pH HIER buffers for many antibodies.  Most of these range from pH 8-9 and might work for your purposes.

Dear Saritha,

The retrieval depends on the antigen and its conformation during cross-linking  and processing, rather than the type of tissue, species and so on.

So for standard testing, according to my experience one can use:

pH6 (Citrate)

pH8(Tris EDTA)

pH9(Tris EDTA)

Proateases ( Prot.K, for Ex)

No retrieval at all (some antigens keep their detectable by an antibody epitopes and these can be destroyed in a retrieval procedure. Just keep that in mind!)

I hope, I helped a bit.

Best regards,

Ivan

What I didn't read in any of these suggestions is to combine it w/ (microwave) heating. If I were you I would try the buffers suggested by Kanchev, above, in combination with 5 - 15 minutes cooled microwave incubation.

von Willebrand Factor (vWF) Antibody Staining Protocol for Immunohistochemistry

Description: von Willebrand Factor (vWF) is a large glycoprotein with a multimeric structure and a molecular mass ranging from 500 kDa up to more than 10,000 kDa, the latter being the largest known for a soluble human plasma protein. The primary product of the von Willebrand Factor gene, located at chromosome 12p13.2, is a 2813 amino acid protein comprising a signal peptide of 22 amino acids, a large propeptide of 741 amino acids, and a mature vWF molecule containing 2050 amino acids. Expression of the von Willebrand Factor gene is tissue specific and confined to endothelial cells and megakaryocytes. vWF is present in plasma, in the Weibel Pallade bodies of endothelial cells, in the alpha-granuel in megakaryocytes and platelets derived from them, as well as in the subendothelial matrix of the vessel wall. Von Willebrand Factor serves as a carrier for Factor VIII in plasma, protecting the circulating coagulation coenzyme from proteolytic degradation. This antibody is suitable for immunohistochemistry and useful tool for studying angiogenesis in neoplasms such as breast cancer and for identification of epithelioid hemangioendothelioma.

Dilution: 1:500 using IHC-TekTM Antibody Diluent  to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60 min/room temperature

Antigen Retrieval

Device: Incubator

Buffer/pH value: IHC-TekTM Epitope Retrieval Solutionor IHC-TekTMProteinase K Solution 

Heat/Cool Temperature: 37 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 (endothelial cell of blood vessels)

Images: Search image

Additional Information:

Species Reactivity: Human, mouse, rat, chicken, cow, dog, sheep and swine

Fixation: Formalin fixed paraffin sections

Positive Control: Tonsil, liver, kidney, skin

Negative Control: Omit primary antibody, isotype control, 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

IHC-TekTM

Immunohistochemistry Reagents

IHC-TekTM series products are specially designed for use in immunohistochemistry (IHC), immunocytochemistry (ICC) and immunofluorescence (IF) staining of tissue sections and cultured cells. The aim is to simplify staining procedure, to enhance staining intensity, and to reduce background. Users can perform high quality immunostaining within short period of time using IHC-TekTM Ready To Use (RTU) reagents.

HC-TekTM Epitope Retrieval Solution, Ready To Use

This newly formulated solution is to be used universally for retrieving antigens and epitopes of all types, including membrane proteins, cytoplasmic proteins as well as nuclear proteins, on formalin fixed paraffin embedded tissue sections during immunohistochemistry procedure.

This solution is superior to commonly used Citrate based solutions and less tissue destruction than other high pH solutions.

IHC-TekTM Epitope Retrieval Steamer Set

This Epitope Retrieval Set utilizes steaming method to achieve antigen unmasking on formalin fixed paraffin embedded tissue sections. The set comes with:

• One B&D steamer

• Six plastic coplin jars with 10 slides capacity of each.

• One bottle of 500 ml IHC-Tek Epitope Retrieval Solution

• One user manual