Dec 06, 2024

Public workspaceEdU detection on tissue sections V.1

DOI
dx.doi.org/10.17504/protocols.io.e6nvwbdrzvmk/v1
  • Ashley Seifert1,
  • Sarah Calve1
  • 1University of Kentucky
Ashley Seifert
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.e6nvwbdrzvmk/v1
Protocol Citation: Ashley Seifert, Sarah Calve 2024. EdU detection on tissue sections. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwbdrzvmk/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: December 06, 2024
Last Modified: December 06, 2024
Protocol Integer ID: 114538
Keywords: ASAPCRN
Abstract
This protocol describes the procedure for EdU detection used by the Seifert Lab. It assumes a starting sample of a paraffin-embedded tissue section and refers to the Seifert Lab protocol for fluorescent immunohistochemistry [add link when published].

It is adapted from the procedure described in A. Salic, T.J. Mitchison, A chemical method for fast and sensitive detection of DNA synthesis in vivo, Proc. Natl. Acad. Sci. U.S.A.
105 (7) 2415-2420, https://doi.org/10.1073/pnas.0712168105 (2008).
Protocol materials
ReagentTris-buffered saline (TBS), 1x solutionFisher ScientificCatalog #BP24721
ReagentHoechst 33342Catalog #H3570
ReagentProlong GoldThermo Fisher ScientificCatalog #P36930
Deparaffinize and rehydrate slides per normal IHC protocol
If using subsequent antibodies for IHC, perform antigen retrieval
Optional
Rinse slides two times in ReagentTris-buffered saline (TBS), 1x solutionFisher ScientificCatalog #BP24721

OPTIONAL
Block for Duration00:05:00 to Duration00:10:00 in appropriate blocking solution
Note
This is not necessary for rodent ear or skin tissue


Optional
Leave slides in TBS until reaction cocktail is ready
Prepare EdU reaction cocktail:

ABC
ComponentVolume (μL)Notes
2 M Tris, pH 8.55100 mL = 24.2 g
50 mM CuSO421 mL = 7.98 mg
0.5 M ascorbic acid201 mL = 88 mg *UNSTABLE AT RT, MAKE FRESH
AlexaFluor Azide (0.25 mg/mL)0.20.5 mg in 2 mL DMSO
ddH2O73
Total Volume:100


Note
  • IMPORTANT: use the reaction cocktail within Duration00:15:00 of preparing
  • Use half the indicated AlexaFluor Azide to reduce background staining (0.1 µL per 100 µL
  • 10 mM EdU = 2.55 mg/mL
  • too much ascorbic acid messes up phalloidin staining
  • 250 nM AlexaFluor azide (AF488) for staining of live cells is 0.45 µL/mL
  • Use half the indicated AlexaFluor Azide to reduce background staining (0.1 µL per 100 µL)






Remove TBS and blot slides
Add reaction cocktail to each slide
Note
Amount100 µL to Amount250 µL usually works well


Incubate for Duration00:30:00 at TemperatureRoom temperature protected from light

Wash slides two times for Duration00:05:00 each in TBS

EITHER:
Continue with normal fluorescent IHC protocol from blocking step
Note
IMPORTANT: keep slides protected from light for all steps to reduce bleaching of EdU signal

Optional
OR:
Counterstain nuclei by incubating with Concentration1 μg/mL ReagentHoechst 33342Contributed by usersCatalog #H3570 for Duration00:05:00

Optional
Rinse 3 times with ddH2O
Mount with ReagentProlong GoldThermo Fisher ScientificCatalog #P36930