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Spectral C1si imaging / linear unmixing tutorial



AIM:

Triple indirect immunostaining of the Mallory hyalin rich hepatocellular carcinoma for p62 component of aggresome, ubiquitin and ubiquitin protein conjugates. Additional nuclear staining with DAPI. Overall, 4 fluorescent labels present in the sample.

MATERIAL:

Formalin fixed paraffin embedded hepatocellular carcinoma rich in Mallory hyalin aggregates

ANTIBODIES:

Primary antibodies:

guinea pig polyclonal anti-p62 component of aggresome(Progen)
mouse monoclonal (clone FK2) anti- mono- and poly ubiquitinated proteins(Biomol)
rabbit polyclonal anti-ubiquitin (DAKO)

Secondary antibodies (Molecular Probes):

Alexa Fluor 405 goat anti-mouse IgG
Alexa Fluor 488 goat anti-rabbit IgG
Alexa Fluor 546 goat anti-guinea pig IgG

DAPI - nuclear stain

MICROSCOPE SETUP:

laser lines - 405 and 488 nm, separate sequential data acquisition
spectral C1 si mode of spectral data acquisition - spectral range 410-720 nm, 32 PMTs, resolution 10 nm
ApoTIRF 60x 1.49 objective
20R/80T beamsplitter

DATA ACQUISITION:

Single labeled samples for reference spectra acquisition. The spectra were acquired from regions of interest (shown) from the spectral confocal images and used for linear unmixing

mono- and poly ubiquitinated proteins + Alexa Fluor 405 (spectral image, measured spectrum, unmixed image)

ubiquitin + Alexa Fluor 488 (spectral image, measured spectrum, unmixed image)

p62 component of aggresome + Alexa Fluor 546 (spectral image, measured spectrum, unmixed image)

DAPI (spectral image, measured spectrum, unmixed image)

Multiple labeled sample (mono- and poly ubiquitinated proteins + Alexa Fluor 405, ubiquitin + Alexa Fluor 488, p62 component of aggresome + Alexa Fluor 546, DAPI). Data acquired sequentially with 405 and 488 laser lines.

405 laser line excitation spectral image (DAPI + Alexa Fluor 405)
488 laser line excitation spectral image (Alexa Fluor 488, Alexa Fluor 546)

Linear unmixing of the images with the reference spectra (original spectral image on the left, reference spectrum in the middle, unmixed image on the right)

Alexa Fluor 405 (mono- and poly ubiquitinated proteins) - magenta
Alexa Fluor 488 (ubiquitin) - green
Alexa Fluor 546 (p62 component of proteasome) - red
DAPI (nuclei) - blue

RESULT:

Merged 4 channel image (red, green, blue, magenta)

Note:
Impact of the use of 488/543/633 dichroic mirror in comparison to 20R/80T beamsplitter demonstrated on the DAPI spectrum (left 488/543/633 DM, right 20R/80T beamsplitter).