Tissue samples were cryo-sectioned on a Leica CM30505 cryostat, set at −24 °C, with a section thickness of ~10 μm. Resulting sections were air dried on to Melinex films and subsequently attached to SEM stubs with silver dags (Agar, UK). They were carbon-coated before being placed in the SEM chamber for analysis. The samples were imaged with a Philips/FEI XL30 field emission gun (FEG) SEM using secondary or back scattered electron detectors as noted in the text. Energy Dispersive X-ray Spectroscopy (X-ray microanalysis) was carried out at 20 kV using an Oxford Instruments ATW SiLi spectrometer running their INCA software.
Cm30505 cryostat
Manufactured by Leica
The Leica CM30505 is a cryostat, a device used for cutting thin, frozen sections of biological samples for microscopic examination. It provides precise temperature control and consistent sectioning capabilities to prepare high-quality samples for analysis.
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Lab products found in correlation
Atw sili spectrometer, by Oxford Instruments (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Rhodamine 123, by Thermo Fisher Scientific (1 mentions)
Mvs10, by Olympus (1 mentions)
Texas red, by Thermo Fisher Scientific (1 mentions)
Evans blue, by Merck Group (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
Lsm 5 pascal, by Zeiss (1 mentions)
Lsm700 axioobserver, by Zeiss (1 mentions)
Fluorogold, by Thermo Fisher Scientific (1 mentions)
Fv300, by Olympus (1 mentions)
6 protocols using cm30505 cryostat
1
Cryosectioning and SEM Analysis
2
Quantifying Blood-Brain Barrier Permeability
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Evan’s blue and fluorescent dye were used as measures for BBB permeability in mice. Evans blue (2% in saline; 4 mL/kg, Sigma, CA), or mixture of Texas Red (6 mg/Kg, Alfa Aesar, MA) and rhodamine-123 (6 mg/Kg, Life Technologies, CA) was intravenously administered through tail vein 30 minutes prior to euthanization. Animals were transcardially perfused with saline and brains were sectioned with a 2 mm brain matrix for Evan’s blue extravasation assay. Hemisphere samples were weighed, homogenized with 400 uL PBS, and precipitated with 50% trichloroacetic acid (Sigma, CA) overnight. All samples were centrifuged at 1000 rpm for 30 minutes to separate out the brain tissue in pellet prior to measuring. Absorption was quantified at 610 nm with a plate reader (BioTek, Winooski, VT). To quantify Evans blue in the brain, an Evans blue standardized curve was used. Results were quantified as microgram/gram brain tissue. For Rhodamine and Texas Red detection, all brains were frozen immediately in −80 °C isopentane. Leica CM30505 Cryostat was used to cut brains slices (20 µm). All brain sections were inspected for fluorescence under a microscope (Olympus MVS10, Japan). Every 5 sections between bregma = 0 mm and bregma = −2 mm were photographed for analysis of mean fluorescence intensity using ImageJ software.
3
Cryosectioning and SEM Analysis
4
Brain Sectioning and Immunostaining in BBS8 Mice
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Mice (n = 3/genotype) were perfused with 4% PFA and brains were removed and cryo-protected in 30% sucrose for 48 h. Following cryo-protection, 10 μm brain sections were mounted on Superfrost glass slides using the Leica CM30505 cryostat. Brain sections were washed in PBS 3 times for 5 min and blocked in PBS containing 0.1% triton X-100, 1% BSA for 1 h. After blocking, slides were incubated with primary antibodies (Table 3 ) overnight at 4 °C. The next day, slides were washed 3 times in PBS for 5 min followed by incubation in the corresponding Alexa Fluor secondary antibody for 1 h. Slides were then washed in PBS 3 times for 5 min and subsequently counter stained for Nuclei with VectaShield containing DAPI (Vector laboratories). Brain sections were matched as closely as possible to their corresponding wildtypes and images were captured with same parameters using confocal microscopy to represent immunoreactivity of various molecular markers associated with reactive astrocytes (Zeiss 710 confocal). For quantifying GFAP and VIMENTIN immunoreactivity from SVZ sub-regions of BBS8 mice (n = 4\genotype), NIH Image J software was used to assess the mean fluorescence intensity of GFAP and VIMENTIN.
5
Fluorescent Microscopy of Plant Tissues
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Fixed and stained tissues were mounted in water prior to microscopy with a Zeiss LSM 5 PASCAL or with a Zeiss LSM 700 AxioObserver system. Fluorescein was excited at 488 nm and detected with a band pass filter of 505–530 nm. Alexa 594 was excited at 555 nm and detected with a 585 long pass filter. GFP was excited at 488 nm and detected with a band pass filter of 505–530 nm. All other microscope settings such as pinhole, laser power, detector gains remained the same for treated versus untreated samples. Autofluorescence of chloroplasts was excited at 488 nm detected with a long pass filter of 560 nm. For DAPI or propidium iodide imaging, tissue was viewed on a Zeiss LSM 510 Meta system with an inverted microscope using a multi-track configuration. DAPI was excited at 405 nm and detected with a band pass filter of 420–480 nm. Propidium iodide was excited at 545 nm and detected with a long pass filter of 585 nm. Seed material was sectioned before imaging by freezing at − 24 °C in Tissue Tek® O.C.T. (optimum cutting temperature) embedding medium, followed by cutting 30 micron sections using a Leica CM30505 cryostat.
6
Retrograde Labeling of DRG Neurons
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L4 and L5 DRG neurons were retrograde labeled with a fluorescent dye, Fluoro-Gold (Molecular Probes, Eugene, OR, USA) at 4 weeks postoperatively. After anesthesia, the crushed sciatic nerves were cut, and Fluoro-Gold was put on the distal side of the proximal part of the sciatic nerve and then sutured. After 5 days, the rats were deeply anesthetized and perfused with saline and paraformaldehyde as described previously (Savignat et al., 2008; Li et al., 2012). The ipsilateral L4 and L5 DRGs were removed and postfixed overnight with paraformaldehyde. DRGs were immersed in a 20% sucrose solution for 4 days, embedded in Tissue Tek (Sakura, Tokyo, Japan), and frozen in liquid nitrogen. Serial 35 μm longitudinal sections were cut at −20°C using a cryostat microtome (CM30505 Cryostat, Leica). Sections were then observed under a fluorescence microscope equipped with a rhodamine filter (Olympus FV-300). The number of Fluoro-Gold-positive neurons in each DRG was counted. The positive-labeled neurons at the DRG section of each group were randomly selected, and their area (soma size of neuron) was measured and averaged using computer software (OPTIMAS Ver. 6.5) (Savignat et al. 2008).
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