Tissue slides are baking 1 hour before dewaxing

Tissue dewaxing is performed with 5 minutes sequential wash in 3 times 100% xylene, 3

times 100% EtOH, followed by 90%, 80%, and 75% EtOH

Tissue slides are then dipped in water several times to finish rehydration

Heat antigen retrieval steps are performed with 300 ul of unmasking solution in 32 ml of H2O in a steamer for 20 minutes

Blocking is performed with Peroxidase and Alkaline Phosphatase Blocking reagent for 15

minutes

Primary antibody is incubated 1 hr at room temperature or 4 C overnight, depending on

targeted protein

Slides are incubated with 1 drop of anti-ms HQ (or anti-rb HQ depends on primary

antibody host animal) at room temperature for optimized time

Slides are incubated with 1 drop of anti-HQ HRP at room temperature for optimized time

TSA dyes are incubated for optimized time

Antibody removal kit or heat mediated antibody stripping is applied to remove the primary antibody

Repeat step 5-10 until all the fluorescent channels are occupied

Counter stain the slides and imaging with fluorescent microscope

Bleaching is performed with 4.5% of H2O2 and 24mM NaOH made up with PBS (CycIF bleaching protocol)

Repeat step 5-13 for different rounds of imaging