IHC was done as previously described (19 (link)). Briefly, 5 μm sections of formalin-fixed paraffin-embedded tissue were de-paraffinized and rehydrated with ethanol. Antigen retrieval was done in water bath heating at 97° with citrate buffer PH6 (Thermofisher). Immune labeling was done using overnight incubation at 4° with anti ITGAX (CD11C) 1:20 dilution (Myebiosource, San Diego, USA) and anti MHCII 1:100 dilution (abcam, Cambridge, USA). Detection of the primary antibody was done with Horeseradish peroxidase (HRP) and Diaminobenzidine (DAB) substrate (Thermofisher). Multiple random photomicrographs were taken for each lymph node in the paracortex region and gingival tissues in the lamina propria, at 40x objective lens using Zeiss microscope (Zeiss AxioIma, Carl Zeiss Microscopy GmbH, Jena, Germany) and quantification was done blindly using automated Image-J software (https://imagej.nih.gov/ij/ ) with a set threshold based on the negative control.
Protocol detail
Immunohistochemical Characterization of Immune Cells
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Antibody
Antigen
Bath
Buffer
Citrate
Dilution
Embedded paraffin
Ethanol
Formalin
Gingival
Lamina propria
Lens
Lymph node
Microscopy
Peroxidase
Photomicrographs
Tissues
Corresponding Organization :
Other organizations : Augusta University, Washington University in St. Louis, William Harvey Research Institute
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Variable analysis
independent variables
- Antigen retrieval method (water bath heating at 97°C with citrate buffer pH 6)
- Quantification of ITGAX (CD11C) and MHCII immune labeling in the paracortex region of lymph nodes and lamina propria of gingival tissues
- 5 μm sections of formalin-fixed paraffin-embedded tissue
- Overnight incubation at 4°C with anti-ITGAX (CD11C) and anti-MHCII antibodies
- Detection of primary antibodies using HRP and DAB substrate
- Multiple random photomicrographs taken at 40x objective lens using Zeiss microscope
- Quantification done blindly using automated Image-J software with a set threshold based on the negative control
- Negative control
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