Table 4.

Technological variables influencing immunohistochemical visualisation.

Step	Variable
Fixation	Time and temperature of cold ischemia before fixation; Choice of the fixative; Time of fixation; underfixation and overfixation
Processing	Protocol of dehydration; Incubation time in xylene and paraffin; Temperature of melted paraffin
Antigen retrieval	Type of antigen retrieval: heat-induced antigen retrieval (HIER) vs. enzymatic treatment vs. none
HIER parameters	Mode: microwave vs. temperature; Temperature, time, pressure (if applicable); pH of the buffer: acidic (e.g., citrate; pH = 6.0), neutral (e.g., TBS; pH = 7.6) vs. basic (e.g., TEG; pH = 9.0)
Primary antibody	Clonality (polyclonal vs. monoclonal), clone, isotype Dilution; Incubation time and temperature
Visualisation system	Choice of the system
Washing of tissue sections	Excessive or insufficient