RNAscope (Advanced Cell Diagnostics, Hayward, California) was used for ultrasensitive detection and visualization of weakly expressed mRNAs in the LS. All mouse-specific probes for A2AR, Slc32a1, and 3-plex negative control probe were synthesized by the manufacturer. Mice were deeply anesthetized with Avertin before quickly removing their brains. After fixing with 4% PFA, brains were cut with a freezing microtome (Leica) into 12 µm sections, adhered to SuperFrost Plus slides (Epredia), and immediately refrozen at −80 °C. Positive (mouse Ppib, Peptidylprolyl Isomerase B) and negative (DapB, 4-hydroxy-tetrahydrodipicolinate reductase) control probes were included in each experiment. The samples were processed according to RNAscope® Multiplex Fluorescent Assay manual. Briefly, sections underwent two steps of pretreatment, including a 10-min step of protease digestion. Hybridization with specific probes was then performed for 2 h at 40 °C, followed by three steps of amplification. Opal fluorescent 520 and 570 were used to detect the chromogen in the exposure step. Two washes of 2 min were observed between each amplification step. Image acquisition was performed with Zeiss LSM 880 NLO confocal microscope.
Superfrost plus slides
Manufactured by Epredia
Sourced in United States
SuperFrost Plus slides are a type of microscope slide designed for histological and cytological applications. They provide a superior adhesion surface to help securely mount and retain samples during processing and staining procedures.
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Lab products found in correlation
Freezing microtome, by Leica (1 mentions)
Lsm 880 nlo confocal microscope, by Zeiss (1 mentions)
Ventana discovery ultra, by Roche (1 mentions)
Hm 325 rotary microtome, by Thermo Fisher Scientific (1 mentions)
5 iodo 2 deoxyuridine, by Merck Group (1 mentions)
Ab6326, by Abcam (1 mentions)
5 chloro 2 deoxyuridine, by Merck Group (1 mentions)
Bmn 673, by MedChemExpress (1 mentions)
Ab13970, by Abcam (1 mentions)
Tcs sp5 2 confocal microscope, by Leica (1 mentions)
Peroxidase streptavidin, by Jackson ImmunoResearch (1 mentions)
Hm355s microtome, by Thermo Fisher Scientific (1 mentions)
Fluorescent secondary antibody, by Jackson ImmunoResearch (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
F7425, by Merck Group (1 mentions)
Ab18465, by Abcam (1 mentions)
Alexa fluor 555 tyramide superboost kit, by Thermo Fisher Scientific (1 mentions)
Tris based solution, by Vector Laboratories (1 mentions)
Nis element, by Nikon (1 mentions)
Mayer s hematoxylin solution, by Merck Group (1 mentions)
6 protocols using superfrost plus slides
1
Ultrasensitive mRNA Visualization in LS
2
Histological Processing of Tumor Samples
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Tumour samples were fixed overnight in 4% paraformaldehyde at 4 °C, dehydrated in graded ethanol baths, cleared with xylene, embedded in paraffin and cut into 4-µm-thick sections using the HM 325 Rotary Microtome (Thermo Fisher Scientific). These sections were mounted onto Superfrost plus slides (Epredia, J1800AMNZ) and allowed to dry for 2 days at room temperature. Haematoxylin and eosin staining was performed at the EPFL Histology Core Facility using the Ventana Discovery Ultra automated slide preparation system (Roche).
3
DNA Replication Dynamics under Stress
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For each LCL sample, ∼100 000 cells were labelled with thymidine analogues, 20 µM 5-chloro-2′-deoxyuridine (CldU, Sigma-Aldrich, C6891) for 30 min followed by 200 µM 5-Iodo-2′-deoxyuridine (IdU, Sigma-Aldrich, I7125) for 30 min and subsequently washed thrice with PBS. Labelled cells were mixed with lysis buffer in 1:1 ratio before mounted on SuperFrost Plus slides (Epredia, J1800AMNZ). Cellular DNA were spread by slanting the slides and fixed in 3:1 methanol/acetic acid for 30 min, followed by denaturation with 2.5 N hydrochloric acid for 40 min and subsequently blocked with 2% BSA in PBS-0.1% Tween20 for 40 min at RT. Slides were washed five times with PBS followed by 2 h incubation with 1:200 anti-BrdU/IdU (Becton Dickinson, 347580, mouse) and 1:200 anti-BrdU/CldU (Abcam, ab6326, rat) antibodies. Subsequently, slides were washed five times with PBS and finally stained with anti-mouse AlexaFluor-488 (Abcam, 150157) and anti-rat AlexaFluor-594 (Abcam, 150116) conjugated secondary antibodies for 1 h at RT.
Labelled DNA fibres were imaged with 63× objective (0.103 um/pixel) using a Zeiss Inverted Fluorescence Live Cell Microscope AO7. The experiments were performed in the presence of replication stress inducing agents, such as HydroxyUrea (HU, Sigma-Aldrich, H8627) and PARP inhibitor (PARPi) (Talazoparib BMN-673, MedChem Express), as controls to assess the differences in RF speed.
Labelled DNA fibres were imaged with 63× objective (0.103 um/pixel) using a Zeiss Inverted Fluorescence Live Cell Microscope AO7. The experiments were performed in the presence of replication stress inducing agents, such as HydroxyUrea (HU, Sigma-Aldrich, H8627) and PARP inhibitor (PARPi) (Talazoparib BMN-673, MedChem Express), as controls to assess the differences in RF speed.
4
Immunohistochemical Analysis of Mouse Brain
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Mouse brains were fixed via immersion in 4% PFA and embedded in paraffin. Next, 7 µm frontal sections were prepared using a HM355S microtome (Thermo, Waltham, MA, USA) and mounted on SuperFrost Plus slides (Epredia, Portsmouth, NH, USA). Antigen retrieval was carried out by boiling the sections in Tris-based solution (Vector Laboratories, Newark, NJ, USA) for 20 min. Sections were blocked with 10% horse serum in PBS containing 0.1% Triton X-100 (0.1% PBTx) for 1 h and incubated with primary antibodies diluted in 5% horse serum in 0.1% PBTx at 4 °C overnight. Sections were incubated with fluorescent secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA), diluted in 5% horse serum in 0.1% PBTx at room temperature for 90 min, and stained with DAPI (Invitrogen). Imaging was performed using a TCS SP5 II confocal microscope (Leica Microsystems, Wetzlar, Germany). For biotin labeling, streptavidin–peroxidase (Jackson ImmunoResearch) was added to primary antibodies and Alexa Fluor 555 Tyramide Super Boost Kit (Invitrogen) was used for signal amplification according to manufacturer’s manual. The following primary antibodies were used: guinea pig anti-Bcl11a [26 (link)], rat anti-Bcl11b (ab18465, Abcam, Cambridge, UK), rabbit anti-FLAG (F7425, Sigma, St. Louis, MO, USA), and chicken anti-GFP (ab13970, Abcam).
5
Histological Quantification of Lipid Emboli
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Tissue samples of the brain, lungs, and heart measuring 1 × 1 × 0.3 cm were frozen in optimal cutting temperature (O.C.T.) compound (Tissue-Tek; Sakura) on dry ice. Tissue slices of 8 µg were cut at −20°C using a Leica CM1950 cryostat (Leica Biosystems). The slices were mounted on Superfrost Plus slides (Epredia) and air-dried, fixed with 4% neutral buffered formalin mixed with 63% ethanol for 5 minutes, dipped in 60% isopropanol for 2 minutes, and incubated in a 0.3% solution of oil red O (Sigma-Aldrich) (6 mL of 0.5% oil red O diluted with 4 mL of H2O) for 10 minutes.
After incubation, samples were rinsed in 60% isopropanol and counterstained with Mayer’s hematoxylin solution (Sigma-Aldrich) for 5 minutes, rinsed for 10 minutes under running tap water, and mounted with glycerol jelly. Images of oil red O-stained samples were captured using a Nikon DS-Fi3 camera (Nikon Systems) installed on a Nikon ECLIPSE Ci light microscope. The images were processed using NIS-Elements (Nikon).
We defined a positive biopsy as ≥2 intravascular or perivascular emboli stained with oil red O in the same section.
After incubation, samples were rinsed in 60% isopropanol and counterstained with Mayer’s hematoxylin solution (Sigma-Aldrich) for 5 minutes, rinsed for 10 minutes under running tap water, and mounted with glycerol jelly. Images of oil red O-stained samples were captured using a Nikon DS-Fi3 camera (Nikon Systems) installed on a Nikon ECLIPSE Ci light microscope. The images were processed using NIS-Elements (Nikon).
We defined a positive biopsy as ≥2 intravascular or perivascular emboli stained with oil red O in the same section.
6
Immunofluorescence Staining of Tissue Sections
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Tissues were dissected immediately post-mortem then washed in ice-cold PBS, fixed for 24 hr (small intestine) or 2 hr (E10.5 embryos) in 4% PFA in PBS, then passed through 10% and 30% sucrose in PBS solutions, and mounted in OCT (Tissue-Tek). 20 µm sections were cut on a Leica CM1850 and adhered to Superfrost Plus slides (Epredia). Sections were post-fixed in 4% PFA in PBS for 10 min at room temperature, then permeablised in 0.5% Triton X-1000 for 5 min at room temperature, and washed twice in 0.2% Triton X-1000 for 5 min at room temperature. Sections were then blocked in 4% BSA in PBS for 1 hr (small intestine) or 2 hr (E10.5 embryo) at room temperature, and primary antibodies were diluted in 4% BSA in PBS and applied overnight at 4°C. Sections were washed three times in 0.2% Triton X-1000, and secondary antibodies were diluted in 4% BSA in PBS and applied at room temperature for 2 hr. Sections were then washed as previously, stained with DAPI at 1 µg/ml, and mounted in Vectashield (Vector Labs). To reduce autofluorescence, staining of small intestine sections also included an additional treatment with 0.1% Sudan Black in 70% ethanol at room temperature for 20 min immediately prior to DAPI stain and mounting.
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