mxIF protocol
Fan Wu
Abstract
This protocol is developed by SenNet JHU TMC group
Steps
Dewaxing and rehydration
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
Heat antigen retrieval steps are performed with 300 ul of unmasking solution in 32 ml of H2O in a steamer for 20 minutes
Blocking
Blocking is performed with Peroxidase and Alkaline Phosphatase Blocking reagent for 15
minutes
Primary antibody incubation
Primary antibody is incubated 1 hr at room temperature or 4 C overnight, depending on
targeted protein
Signal amplification
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
Antibody removal kit or heat mediated antibody stripping is applied to remove the primary antibody
Multiplex IF
Repeat step 5-10 until all the fluorescent channels are occupied
Counter stain the slides and imaging with fluorescent microscope
Bleaching
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
