Apr 19, 2024

Public workspaceImmPRESS Polymer Detection Following BaseScope In Situ Hybridization

DOI
dx.doi.org/10.17504/protocols.io.kqdg325rzv25/v1
  • 1Van Andel Research Institute
madalynn.erb Erb
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DOI: dx.doi.org/10.17504/protocols.io.kqdg325rzv25/v1
Protocol Citation: madalynn.erb Erb 2024. ImmPRESS Polymer Detection Following BaseScope In Situ Hybridization. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg325rzv25/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: April 19, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 98492
Keywords: ASAPCRN
Funders Acknowledgements:
Madalynn Erb
Grant ID: ASAP-000592
Abstract
Protocol for performing ImmPRESS immunostaining with alkaline phosphatase labeling after BaseScope In Situ Hybridization mouse brain sections.
Materials
Reagents: ImmPRESS‱-AP Horse Anti-Rabbit IgG Polymer Detection Kit (MP-5401; Vector Labs) Vector‱ Blue
Alkaline Phosphatase Substrate (SK-5300; Vector Labs)
Day 1
Day 1
Complete BaseScope in situ hybridization using 35um mouse brain sections. Do not dry slides.
For BaseScope protocol see : (dx.doi.org/10.17504/protocols.io.5qpvo364zv4o/v1)
Wash slides 2 times in PBS at room temp 5 minutes each wash
Block sections in 0.4% Bovine Serum Albumin (BSA), 10% horse serum, 0.3% triton X-100 for 1 hour at room temp
Incubate sections in rabbit anti TH (1:500) in 0.4% BSA, 5% horse serum and 0.1% tritonX-100 overnight at 4 degrees C
Day 2
Day 2
Wash slides in PBS at room temp (3 times 5 minutes each wash)
Incubate slides in secondary antibody (ImmPRESS-AP Polymer Reagent) at room temp for 2 hours.
Wash slides in PBS at room temp (3 times 5 minutes each wash)
  1. Prepare the Alkaline Phosphatase Substrate

a. Make 2.5mL 100mM Tris-HCl, pH 8.2
b. Add 1 drop of Reagent 1
c. Add 1 drop of Reagent 2
d. Add 1 drop of Reagent 3
e. Mix well – use immediately
Incubate sections in alkaline phosphatase substrate for 5 minutes
Wash slides in PBS (2 times 5 min each wash)
Dry slides at 60°C for ≥15min until they are completely dry
Coverslip slides using VectaMount
Store slides at room temp
Protocol references

Preparation of Free Floating Coronal Mouse Brain Sections : dx.doi.org/10.17504/protocols.io.dm6gp3pb8vzp/v1

BaseScope In Situ Hybridization: (dx.doi.org/10.17504/protocols.io.5qpvo364zv4o/v1)