Protocol refinement for quenching autofluorescence of red blood cells in FFPE sections of organ samples from cattle, pigs and chickens

Abstract

One of the most common problem that researchers encounter when using fluorescence to visualize immunohistochemistry is the autofluorescence of the studied organ tissue sections and cell cultures. Autofluorescence quenching is necessary for a wide variety of organs and tissues, as well as for different methods of fixation and histochemical processing of sections. In addition to autofluorescence quenching, it is necessary to take into account the need for histological readability of tissue sections when using counterstains afterwards. Such protocol refinement for fluorescent immunohistochemistry for chicken, porcine and cattle tissues was carried out for the first time, as well as the use of a dimethyl sulfoxide (DMSO) solution with ethanol as Sudan Black B (SBB) solvent. Incubation of sections in SBB was chosen as the simplest and most nonspecific one. The most effective dissolution of the dye is achieved at a concentration of 0.3% SBB in a solution of 70% ethanol and absolutized DMSO in a 4:1 v/v ratio. The most thorough removal of SBB solution excess is achieved by rinsing the sections 5 times with 70% ethanol and then rinsing the sections with TBST (tris-buffered saline and Tween-20) buffer 5 times.