Mar 20, 2024
mxIF protocol
- 1Johns Hopkins University
Protocol Citation: Fan Wu 2024. mxIF protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1pb9lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 19, 2024
Last Modified: March 20, 2024
Protocol Integer ID: 96962
Funders Acknowledgement:
Pei-Hsun Wu
Grant ID: UG3 CA275681-01
Abstract
This protocol is developed by SenNet JHU TMC group
Dewaxing and rehydration
Dewaxing and rehydration
2h 5m
Tissue slides are baking 1 hour before dewaxing
1h
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
1h
Tissue slides are then dipped in water several times to finish rehydration
5m
Heat antigen retrieval
Heat antigen retrieval
1h
Heat antigen retrieval steps are performed with 300 ul of unmasking solution in 32 ml of H2O in a steamer for 20 minutes
40m
Blocking
Blocking
30m
Blocking is performed with Peroxidase and Alkaline Phosphatase Blocking reagent for 15
minutes
30m
Primary antibody incubation
Primary antibody incubation
Primary antibody is incubated 1 hr at room temperature or 4 C overnight, depending on
targeted protein
Signal amplification
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
Antibody removal kit or heat mediated antibody stripping is applied to remove the primary antibody
Multiplex IF
Multiplex IF
Repeat step 5-10 until all the fluorescent channels are occupied
Counter stain the slides and imaging with fluorescent microscope
Bleaching
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