Tests

RNAscope Multiplex fluorescence assay (ACDbio)

The test was done on fixed frozen mouse brain in collaboration with Olexiy Kochubey from Prof. Schneggenburger’s lab.

Detection of mRNA and protein on the same slide:

 

Ppib mRNA (green) / Positive control

dtomato protein (red)

Dapb mRNA (green) / negative control

dtomato protein (red)

     

            Oxtr4 mRNA (green) / experimental gene           

            dtomato protein (red)       

         

 

Detection of two mRNAs on the same slide

  

Ppib mRNA (red)

Oxtr4 mRNA (green)

 

 

 

 

 

Double immunofluorescence with primary of the same specie

Double immunofluorescence :

– rabbit anti Ki67 revealed with TSA Alexa568

– rabbit anti CD3 revealed with donkey anti rabbit Alexa488

– DAPI

 Technique based on the paper: Zsuzanna E. Tóth and Eva Mezey, “Simultaneous visualisation of multiple antigens with tyramid signal amplification using antibodies of the same species”, Journal of Histochemistry & Cytochemistry, volume 55(6): 545-554, 2007

 

 

 

 

Inflammatory markers

A panel of different markers for immune cells was tested on paraffin sections form PFA fixed mouse tissue:

– CD45

– CD3

– CD4

– CD8

– B220

– F4/80

Please write an email if you need the references and protocols for those antibodies.

MSB stain for fibrin

The stain is now establish at the facility on paraffin sections.

Cell proliferation

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Test EdU (Invitrogen)

Mouse injected  i.p. with 200ug of EdU 2 hours before sacrifice.
Intestine paraffin sections:
EdU: Azide Alexa594 revelation (Red)
DAPI: nuclei (Blue)
Scale: 20um.

No DNA denaturation is required for EdU revelation compared to BrdU. It is actually a chemical reaction with an azide and not an immunofluorescence based on antibody reaction. Futhermore, it lasts only 2 hours.