Aortic and left ventricular heart segments of SERCA2a/b and SERCA2b/b mice were collected immediately after dissection, embedded in NEG-50TM (Thermo Scientific) and kept at −80°C. Cryosections of the aortic segments (± 6 μm) were air-dried for 20 min and, then, fixated with acetone (5 min). The cryostat sections were rinsed with phosphate buffered saline (PBS, 5 min) at room temperature. Following incubation overnight at 4°C with the primary antibodies against SERCA2a (1:10000, R15-7, homemade), SERCA2b (1:10000, R5-24, homemade) and 3 times wash (10 min) with PBS, they were incubated for 2 h at 37°C with secondary antibody (goat anti-rabbit Alexa green, Molecular Probes). The cryostat sections were placed again in washing buffer (15 min), washed 3 times (5 min) in PBS and mounted in VECTASHIELD Antifade Mounting Medium with DAPI (nuclear staining). Images were made on an Olympus BX40 epifluorescence microscope with a SensiCam 12 bit cooled CCD (PCO, Germany) and stored on computer for later analysis with Photoshop software.
Neg 50tm
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Neg-50TM is a compact and efficient cryogenic freezer designed for reliable long-term sample storage. It utilizes a reliable compressor-based cooling system to maintain temperatures as low as -50°C, providing a secure environment for preserving a variety of samples, including biological specimens, chemicals, and pharmaceuticals.
Automatically generated - may contain errors
Lab products found in correlation
Ms 222, by Merck Group (3 mentions)
Superfrost slide, by Thermo Fisher Scientific (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Vt1000, by Leica (1 mentions)
Citifluor af1, by Agar Scientific (1 mentions)
Phosphate buffered saline pbs, by Eurobio Scientific (1 mentions)
Cryostar nx70, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Cryostat, by Leica (1 mentions)
Bz x810 all in one fluorescence microscope, by Keyence (1 mentions)
Microm hm550, by Thermo Fisher Scientific (1 mentions)
Fsx100 bio imaging navigator, by Olympus (1 mentions)
8 protocols using neg 50tm
1
Quantifying SERCA2a and SERCA2b in Mouse Hearts
2
Guinea Pig Brain Tissue Preparation for Microscopy
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After they were anesthetized with a mixture of ketamine (60 mg/kg), xylazine (10 mg/kg) and acepromazine maleate (10 mg/kg), guinea pigs were perfused transcardially with 0.9% saline and then with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) (n = 2) or Bouin’s fixative (n = 12) for light microscopy, or 2% PFA/2.5% glutaraldehyde in 0.1 M PB (n = 4) for electron microscopy. Brains were removed and post-fixed by immersing in the same fixative overnight at 4 °C. Bouin-fixed tissues were embedded in paraffin and cut into sequential 7 μm frontal sections. PFA-fixed tissues were cryo-protected by immersion in 30% sucrose for 24 h and then embedded in NEG50TM (Thermo-Scientific, Waltham, MA, USA) to cut 50 μm frontal sections using a cryostat (MICROM HM520, Walldorf, Germany). PFA/glutaraldehyde-fixed tissues were cut into 150 μm frontal sections using a vibratome (Leica VT 1000S, Deer Park, IL, USA). For histological and immunohistochemical analysis, the tissues from 14 animals were analyzed, and tissues from four animals were analyzed for electron microscopy (Table S1 ).
3
Vascular Bundle Network Visualization
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For vascular bundle network studies among the whole plant, of the main stem on a 45–50 DAG plants were harvested and fixed in 4% paraformaldehyde (Sigma-Aldrich) in 1 x Phosphate Buffered Saline (PBS, Eurobio), buffer-0,1% tritonX100 (Bioprobe) under vacuum on ice for 2 h. Samples were incubated at 4 °C O/N in the fixative and then washed in PBS and store at 4 °C until use. Thirty μm section were obtain with a HM 650 V Vibratome from MicroMicrotech France. For 3D reconstruction of the node, the 3 cm of the fifth node including upper and lower internode of the main WT stem were fixed as described above and later incubated in 10%, then 20% sucrose for 1 h each, and 30% sucrose overnight at 4 °C. After removing the excess of sucrose, samples were embedded in cryo-embedding medium (NEG-50TM, Thermo scientific) and freeze with liquid nitrogen. Tissue cutting were performed at − 16 °C (CryostarTM NX70, Thermo scientific) and 100 μm section were placed on superfrost slides (Thermo scientific) and stored at − 20 °C or mounted in citifluor AF1 (agar scientific) for confocal imaging.
4
Validating Lpar1-EGFP Expression in Mice
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Mutant mice were identified and validated by standard genotyping procedures. Forward 5′-ACATGGT show [QJ]CCTGCTGGAGTTC-3′ plus Lpar1 Reverse 5′-GAGTGTCCTCATCTCCTCTG-3′) of the neomycin. cryostat sections were prepared from homozygote, heterozygote, and wildtype. Expression of the Lpar1-EGFP fusion allele was assessed by isolating embryonic day 13.5 (E13.5) heads and adult brain and lumbar spinal cord from wildtype and Lpar1-EGFP knock-in age-matched animals, which were processed for standard cryostat sectioning, fixation, and fluorescence microscopy [68 (link)–73 (link)]. In brief, samples were mounted in pre-chilled molds containing Neg-50TM (Thermo Scientific) frozen section media and then frozen on dry ice. Tissue sections were cut at 20 μm using a Leica cryostat, fixed with cold 4% paraformaldehyde in phosphate-buffered saline (PBS) for 5 min, washed twice with PBS, mounted, and coverslipped using VECTASHIELD® HardSetTM antifade mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Tissue section, staining, and mounting was performed at the SBP histology core facility. Endogenous EGFP fluorescence in all tissues was evaluated using a KEYENCE BZ-X810 all-in-one fluorescence microscope. RNAscope [74 (link)] against mouse Lpar1 utilized commercially available probes and protocols (Advanced Cell Diagnostics, Newark, CA).
5
Fixation and Sectioning of Elasmobranch Embryos
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Embryos up to stage 32 were anesthetized with 0.5% tricainemetanesulfonate (MS-222; Sigma, St. Louis, MO, USA) in seawater and separated from the yolk before fixation in 4% paraformaldehyde (PFA) in elasmobranch’s phosphate buffer [EPB: 0.1 M phosphate buffer (PB) containing 1.75% urea, pH 7.4] for 24–48 h, depending on the stage of development. Embryos from stage 33 onwards, juveniles, and the adult were deeply anesthetized with MSS-222 and then perfused intracardially with elasmobranch’s Ringer solution (see Ferreiro-Galve et al., 2008 (link)) followed by PFA 4% in EPB. The eyes of PH (stages 33 and 34) embryos, juveniles, and the adult were removed and postfixed in PFA 4%. After that, the eyes were rinsed in phosphate buffer saline (PBS), cryoprotected with 30% sucrose in PB, embedded in NEG 50TM (Thermo Scientific, Kalamazoo, MI, USA), frozen with liquid nitrogen-cooled isopentane and cut on a cryostat. Parallel series of transverse and sagittal sections (16–18 μm thick) were mounted on Superfrost Plus slides (Menzel-Glässer®, Madison, WI, USA).
6
Histological Evaluation of AAV5-Mediated Gene Delivery
7
Cryoprotected Tissue Sectioning of Zebrafish
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Animals were deeply anesthetised with 0.0016% tricaine methanesulfonate (MS-222, Sigma-Aldrich, St. Louis, MO, USA), euthanised, and fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate-buffered saline pH 7.4 (PBS) for 2 h (from 2 to 7 dpf) and 1 day (from 1.5 mpf to 3–4 ypf) at 4 °C. After fixation, the lens was removed from the eye in specimens from 1.5 mpf onwards. Eyes were dissected out from the rest of the body in specimens from 8.5 mpf onwards. After rinsing in PBS, the animals or eyes were cryoprotected with 30 % sucrose in PBS, embedded in Neg-50TM (Thermo Scientific, Kalamazoo, MI, USA), and frozen with liquid nitrogen-cooled isopentane. Transverse sections (18 μm thick) were obtained on a cryostat and mounted on Superfrost Plus slides (Menzel-Glasser, Madison, WI, USA).
8
Zebrafish Eye Tissue Preparation
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Animals were deeply anesthetized with 0.0016% tricaine methanesulfonate (MS-222, Sigma-Aldrich, St. Louis, MO), euthanized and fixed by immersion in 4% paraformaldehyde in 0.1M phosphatebuffered saline pH 7.4 (PBS) for 2 h (from 2 dpf to 7 dpf) and 1 day (from 1.5 mpf to 3-4 ypf) at 4 °C. After fixation, the lens was removed from the eye in specimens from 1.5 mpf onwards. Eyes were dissected out from the rest of the body in specimens from 8.5 mpf onwards. After rinsing in PBS, the animals or eyes were cryoprotected with 30 % sucrose in PBS, embedded in Neg-50TM (Thermo Scientific, Kalamazoo, MI) and frozen with liquid nitrogen-cooled isopentane. Transverse sections (18 μm thick) were obtained on a cryostat and mounted on Superfrost Plus slides (Menzel-Glasser, Madison, WI).
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