Synthetic controls for immunohistochemistry
Provided herein are methods for generating a solid antigen/carrier protein gel for immunohistochemistry (IHC) staining, as well as gels, kits and methods of use thereto. In particular, the methods, gels and kits provided herein include a purified antigen such as a polypeptide antigen, and a carrier protein such as a serum albumin protein, an egg white protein or mixture of egg white proteins, gelatin, or poly-lysine. Examples are provided in which the purified antigen is cross-linked to the carrier protein in the solid antigen/carrier protein gel.
1. A method for generating a solid antigen/carrier protein gel for immunohistochemical (IHC) staining, the method comprising:
(a) mixing a purified antigen with a liquid solution comprising a carrier protein to produce an antigen/carrier protein liquid solution;
(b) cross-linking the antigen with the carrier protein using a cysteine-reactive reagent; and
(c) heating the antigen/carrier protein liquid solution to form the solid antigen/carrier protein gel,
wherein the antigen is a polypeptide antigen comprising a C-terminal cysteine,
wherein the carrier protein consists of a serum albumin protein, and
wherein the antigen/carrier protein liquid solution further comprises formaldehyde.
2. The method of claim 1 , further comprising, after (b): dehydrating the solid antigen/carrier protein gel and embedding the dehydrated solid antigen/carrier protein gel in a paraffin block.
3. The method of claim 1 , further comprising, after (b): incubating the solid antigen/carrier protein gel in a liquid embedding medium and freezing the solid antigen/carrier protein gel in the embedding medium.
4. The method of claim 1 , further comprising, after (b): embedding the solid antigen/carrier protein gel in a plastic resin.
5. The method of claim 1 , wherein the antigen/carrier protein liquid solution comprises formaldehyde at a final concentration of at least about 1%.
6. The method of claim 1 , further comprising:
after (b), dehydrating the solid antigen/carrier protein gel;
embedding the dehydrated solid antigen/carrier protein gel in a paraffin block;
sectioning the paraffin block with the embedded antigen/carrier protein gel into one or more sections having a thickness of between about 30 nm and about 50 μm;
subjecting the one or more sections of the embedded solid antigen/carrier protein gel to antigen retrieval; and
after antigen retrieval, blocking the one or more sections of the embedded solid antigen/carrier protein gel.
7. The method of claim 1 , further comprising:
after (b), incubating the solid antigen/carrier protein gel in a liquid embedding medium;
freezing the solid antigen/carrier protein gel in the embedding medium;
sectioning the frozen antigen/carrier protein gel into one or more sections having a thickness of between about 30 nm and about 50 μm; and
blocking the one or more sections of the frozen antigen/carrier protein gel.
8. The method of claim 1 , wherein the antigen further comprises an N-terminal tyrosine.
9. The method of claim 1 , wherein the serum albumin protein is bovine serum albumin.
10. The method of claim 1 , wherein the antigen/carrier protein liquid solution produced in (a) comprises the carrier protein at a concentration of greater than or equal to 2% (w/v) and less than or equal to 25% (w/v).