Histology

The histology platform has currently all the equipment necessary to perform routine
histological techniques on fixed, frozen or fresh samples, including the possibility
of virtualizing your histological slides. The platform also has specific equipment
for the realization of 3D marked sample reconstructions (OHREM), laser microdissection projects and multiplex staining (Phenocycler-Fusion).

Our mission is to take care of your samples according to your request (embedding tissues
in paraffin, cutting tissue, standard or specific staining and scan of your slides in brightfield
in fluorescence, or polarized, HREM) and to train users on different histological techniques (embedding tissues in paraffin, multiplex staining) and on our various workstations available for reservation (microtome, cryostat, vibratome, laser microdissector, PhenoImager).

We offer you our experience and technology for this platform.

The platform is open to external research teams from public and private institutions.

We will be happy to help you with specific and routine analyzes in your research projects.

1. For current service requests

> please complete the dedicated forms and send them by email to histo.sfrnecker@inserm.fr

2. For any requests regarding the possibility of innovative technological developments
or collaborative analyzes

> please contact the head of the platform Sophie Berissi by email sophie.berissi@inserm.fr
(cc scientist referent Dr Ganna Panasyuk at ganna.panasyuk@inserm.fr)

SERVICES

ROUTINE SERVICES

The platform takes care of your samples and performs the services of your choice:

impregnation and embedding of tissues in paraffin

section: fixed or fresh frozen tissue (5-30µm) and / or embedded-paraffin tissue (4µm)

histological staining: standard staining (Hematoxylin - Eosin, Toluidine blue)
or collagen Masson's trichrome, Picro-sirius), polysaccharides (Periodic Acid Schiff) specific staining

the virtualization of slides in brightfield, fluorescence and polarized.

impregnation and embedding of tissues in resin for the section with the OHREM

the sections of sample included in a resin and acquisition of images with the OHREM.

TRAINING OR GUIDED SERVICES

The platform also provides training for one or more members of your team:

the use of laboratory equipment: paraffin embedding, microtome, cryostat, vibratome and PhenoImager.
multiplex staining on the Phenocycler-Fusion (training and support).
laser microdissection (training and support).

MAKE A SERVICE/TRAINING REQUEST

For any service and/or training request, you must contact us by email
at histo.sfrnecker@inserm.fr and send the dedicated forms.

To access the various services, it is imperative to fully subscribe to the platform charter
of use.

Each new user or project requires a meeting to discuss your project, offer you the different services adapted to your needs, explain how our working method and the histological constraints.

EQUIPMENT

Equipment for routine histological technics

TREATMENT OF FIXED TISSUES (FFPE)

1 fully enclosed vacuum tissue infiltration machine tissue processor (ASP300S, Leica) allowing automatic dehydration and paraffin impregnation to be carried out for
up to 300 samples per day.

2 tissue embedding centers (EC-350, Microm France and Histostar, Thermo) to embed the tissues with paraffin

1 cassette dewaxer (TR 302, Tech-Inter) to remove excess paraffin around
the completed block.

SECTIONS

4 microtomes, 2 automatics (2 x HM 355S, Microm France), 1 semi-automatic
(HM 340E, Microm France) for performing paraffin cutting services, as well as
1 semi-automatic microtome (RM2245, Leica) available for users.

1 laser slide printer (HistoCore Lightning S, Leica) for writing slides

1 cryostat (CM3050S Leica) for frozen sections. This device is also accessible
to autonomous users.

1 vibratome (VT1200) for fresh tissue sections. This equipment is also accessible
to autonomous users.

STAINING

1 multistainer (ST5020-CV5030, Leica) for performing histological standard or specific staining classic and specific tissue stains and for mounting with coverslip

1 microscope (DME, Leica) allowing constant control of the quality of sections
and staining.

SLIDE SCANNERS

The facility is equipped with two whole-slide scanners, each with a capacity of 210 slides.

NanoZoomer 2.0-HT (Hamamatsu)
- Imaging modes: Brightfield and fluorescence (DAPI, FITC, TRITC, Cy5)
- Objective: 20×, NA 0.75, providing a total magnification of 20× or 40×

VS200 (Olympus–Evident)
- Imaging modes: Brightfield, fluorescence (DAPI, FITC, Cy3, Cy5, Cy7), and polarized light imaging for collagen detection using Picro Sirius Red staining.
- Objectives: 2×, 4× (NA 0.13), APO 20× (NA 0.80), and APO 40× (NA 0.95)
- Cameras: one color CMOS camera for brightfield imaging and one highly sensitive ORCA-Fusion monochrome camera for fluorescence imaging.

PhenoImager (Akoya), capable of scanning up to 4 slides simultaneously for fluorescence imaging of OPAL-stained slides.
- Objectives: 20× and 40×.

Equipment for specific techniques

LASER MICRODISSECTOR

The platform has also a Laser Microdissector (Palm MicroBeam, Zeiss). It allows to study nucleic acids (DNA / RNA) and proteins of specific cell subpopulations.

Brightfield imaging camera: AxioCam ICc1 rev.3
- CCD 1392x1040= 1,4 Mega Pixels

Fluorescence imaging camera: AxioCam MRm rev3
- CCD 1392x1040= 1,4 Mega Pixels
- Filtres : DAPI (EX 390-420, LP450), FITC/GFP (EX BP 450-490, EM BP 515-565), Rhodamine (EX BP 510-560, EM LP 590)

Objectives : 5x/0,25 ; 20x/0,4 ; 40x/0,6 ; 63x/0,75

OHREM

The histology platform has an OHREM (Optical High Resolution Episcopic Microscope, Indigo Scientific).

HREM is a 3D histology technique. It is useful for analyzing and quantifying the fine anatomy of thick and large samples and can also provide 3D LacZ and HRP reporter images from transgenic samples or ISH experiments. It is not compatible with fluorescent reporter gene, it does not achieve subcellular resolution; it is therefore complementary to confocal or light sheet microscopes, which are often limited in the size of samples that can be processed and are not compatible with non-fluorescent reporters.

Useful link: https://www.indigoscientific.com/hrem

2 light sources: 585nm (enzymatic labeling) or 470nm (morphology)
Cutting thickness: 1 to 5µm
Sample size: up to 20mm
Resolution: 4 or 12 megapixel
Cutting time: 2 to 12 hours (depends on sample size and resolution).

1 binocular magnifier (Stemi 508, Zeiss) for orientation of the samples during resin embedding for the HREM technique.

MULTIPLEX STAINING

The facility has acquired the Phenocycler-Fusion (Akoya), enabling the simultaneous detection of multiple biomarkers for spatial tissue analysis, thereby facilitating the study of cellular organization within their microenvironment.

40+ biomarkers
Staining area: 18 × 34 mm
Tissue thickness: 5–10 µm
Compatible with FFPE and fresh-frozen (FF) samples.

Data analysis is performed in collaboration with Nicolas Goudin's Bio-image analysis facility and the bioinformatics platform.

STAFF

Sophie BERISSI

Head

sophie.berissi@inserm.fr

Ganna PANASYUK

Scientific Referent

ganna.panasyuk@inserm.fr

PUBLICATIONS

Haemodynamic stress-induced breaches of the arterial intima trigger inflammation
and drive atherogenesis.

Franck G, Even G, Gautier A, Salinas M, Loste A, Procopio E, Gaston AT, Morvan M,
Dupont S, Deschildre C, Berissi S, Laschet J, Nataf P, Nicoletti A, Michel JB, Caligiuri G.

Eur Heart J. 2018 Dec 11.

MITF-A controls branching morphogenesis and nephron endowment.

Phelep A, Laouari D, Bharti K, Burtin M, Tammaccaro S, Garbay S, Nguyen C, Vasseur F, Blanc T, Berissi S, Langa- Vives F, Fischer E, Druilhe A, Arnheiter H, Friedlander G,
Pontoglio M, Terzi F.

PLOS Genet. 2017 Dec 14;13(12).

Alternative splicing-regulated protein of hepatitis B virus hacks the TNF --
stimulated signaling pathways and limits the extent of liver inflammation.

Pol JG, Lekbaby B, Redelsperger F, Klamer S, Mandouri Y, Ahodantin J, Bieche I,
Lefevre M, Souque P, Charneau P, Gadessaud N, Kremsdorf D,Soussan P.

FASEB J. 2015 May;29(5):1879-89.

Cystic gene dosage influences kidney lesions after nephron reduction.

Canaud G, Bienaimé F, Viau A, Treins C, Baron W, Nguyen C, Burtin M, Berissi S,
Giannakakis K, Muda AO, Zschiedrich S, Huber TB, Friedlander G, Legendre C, Pontoglio M, Pende M, Terzi F.

Nat Med. 2013 Oct;19(10):1288-96.

AKT2 is essential to maintain podocyte viability and function during chronic
kidney disease.

Canaud G, Bienaimé F, Viau A, Treins C, Baron W, Nguyen C, Burtin M, Berissi S,
Giannakakis K, Muda AO, Zschiedrich S, Huber TB, Friedlander G, Legendre C, Pontoglio M, Pende M, Terzi F.

Nat Med. 2013 Oct;19(10):1288-96.

Antiviral activity of Bay 41-4109 on hepatitis B virus in humanized Alb-uPA/SCID mice. Brezillon N, Brunelle MN, Massinet H, Giang E, Lamant C, DaSilva L, Berissi S, Belghiti J, Hannoun L, Puerstinger G, Wimmer E, Neyts J, Hantz O, Soussan P, Morosan S, Kremsdorf D.

PLoS One. 2011;6(12):e25096

Constitutively active Akt1 expression in mouse pancreas requires S6 kinase 1
for insulinoma formation.

Alliouachene S, Tuttle RL, Boumard S, Lapointe T, Berissi S, Germain S, Jaubert F, Tosh D, Birnbaum MJ, Pende M.

J Clin Invest. 2008 Nov;118(11):3629-38.

HISTOLOGY