Procedures for immunohistochemistry (IHC) are previously described (20 (link)–22 (link)). Briefly, paraffin was removed and tissue was subjected to high pressure antigen retrieval (Vector Unmasking Solution; Vector Laboratories, Burlingame, CA). Samples were incubated in primary antibody (PHGDH, Cell Signaling) overnight at 4°C. Secondary antibodies, raised in donkey (Jackson Immunoresearch, West Grove, PA), were used at a dilution of 1:250 at room temperature for 1-hour. Vectastain reagents and diaminobenzidine (DAB) (Vector Laboratories) were used to develop IHC. Images were subsequently taken on a Zeiss Axio Observer Z1 microscope. Tissue samples from human patients were obtained with patient consent and approval from the University of Utah Institutional Review Board.
Protocol detail
Immunohistochemistry for PHGDH Protein
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Vector Unmasking Solution
PHGDH
Vectastain reagents
diaminobenzidine (DAB)
Axio Observer Z1 microscope
Antibodies
Antibody
Antigen
Dilution
Donkey
Human
Immunohistochemistry
Institutional review board
Microscope
Paraffin
Patient
Pressure
Tissue
Vector
Corresponding Organization : Howard Hughes Medical Institute
Other organizations : Huntsman Cancer Institute, Nationwide Children's Hospital
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Variable analysis
independent variables
- Antigen retrieval method (high pressure)
- Primary antibody (PHGDH, Cell Signaling)
- Secondary antibody (raised in donkey, Jackson Immunoresearch)
- IHC staining intensity and patterns
- Tissue samples from human patients
- Incubation conditions (overnight at 4°C for primary antibody, 1-hour at room temperature for secondary antibody)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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