Fresh monkey anterior eye segments were embedded in OCT and immediately frozen on dry ice prior to storage at −80°C. Twelve-micrometer-thick frozen sections were transferred to glass polyethylene naphthalate foil slides (#11505189; Leica Microsystems, Wetzlar, Germany) as we previously described30 (link) and stained with Eosin Y. TM was dissected using a laser micro-dissection system (LMD 6000 and CTR 6500, Leica Microsystems) (Fig. 3 ). Some areas of uveal meshwork were included to obtain adequate RNA quantities. RNA extraction was performed using the RNeasy Micro Kit (Qiagen, Hilden, Germany) and amplification with Ovation Pico WTA System V2 (NuGEN Technologies, San Carlos, CA, USA). The quantitative PCR (qPCR) analysis was performed using LightCycler 480 Software according to manufacturer protocols. Quantified values for each gene of interest were normalized against the input determined by the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Actb, NM_007393). We custom designed the primers for the monkey species to detect fibronectin, type IV collagen, and NF-κB (Supplementary Table S1 ). Each assay was performed in triplicate utilizing mean values for statistical analysis.
Lmd6000
Manufactured by Leica Microsystems
Sourced in Germany
The LMD6000 is a laser microdissection system designed for precise and efficient sample preparation. It enables the user to accurately isolate specific regions of interest from a variety of sample types, including tissue sections and cell cultures. The system utilizes a high-precision laser to cut and capture target samples for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy micro kit, by Qiagen (2 mentions)
Ovation pico wta system v2, by Tecan (1 mentions)
Ctr6500, by Leica Microsystems (1 mentions)
Blood tissue kit, by Qiagen (1 mentions)
Ab6994, by Abcam (1 mentions)
Ab53554, by Abcam (1 mentions)
Ab133357, by Abcam (1 mentions)
Ab58779, by Abcam (1 mentions)
Nbp1 45057, by Novus Biologicals (1 mentions)
Aida version 4, by Raytest (1 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (1 mentions)
Picopure protocol, by Arcturus (1 mentions)
Recoverall total nucleic acid isolation kit, by Thermo Fisher Scientific (1 mentions)
P2308, by Merck Group (1 mentions)
Genechip human genome u133 plus 2, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (1 mentions)
Fluidic station 450, by Thermo Fisher Scientific (1 mentions)
Messageamp 2 biotin enhanced kit, by Thermo Fisher Scientific (1 mentions)
Cm1950 cryostat, by Leica Microsystems (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
19 protocols using lmd6000
1
Molecular Analysis of Primate Trabecular Meshwork
2
Laser Microdissection of Liver Nodules
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We selected the corresponding paraffin block with DN, specially GPC3-positive DN, according to the results of immunohistochemical staining, and then prepared eight serial 10-µm tissue sections which were then placed on a UV-absorbing membrane for laser microdissection by LMD6000 (Leica Microsystems Ltd, Wetzlar, Germany). After HE staining, the slides were mounted on a microstat, and the selected nodules were dissected by a UV laser through motorized optical beam scanning. The dissected tissues (with the attached specimen ) were pooled into the cap of a 0.5-ml microcentrifuge tube that was filled with 40 µL lysate buffer and 10 µL protease K. Along with each dissected nodule, the surrounding normal liver tissue of the same size was isolated and analyzed as a control. The microcentrifuge tubes were placed in a waterbath (48°C) to digest the tissue specimens. After digestion for 12–20 h, genomic DNA was extracted using Qiagen blood & tissue Kit (Germany) and confirmed by gel electrophoresis (20 g/L agarose ), and then stored at −20°C until use.
3
Multimodal Assessment of Brain Pathology
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Cryosections of the brains were stained with DAPI (4′,6-diamidino-2-phenylindole) and evaluated by fluorescence microscopy. Disturbance of the BBB was visually evaluated using Evans blue dye (EBD). Therefore, 500 μl/kg 2% EBD was injected i.v. 30 min before decapitation of the rat for AR. EBD extravasation in brain slices was examined by fluorescence microscopy (LMD6000, Leica Microsystems CMS GmbH) and processed by AIDA software (AIDA Version 4.50, Raytest).
Blood vessels were stained with anti-rat von Willebrand factor antibody (ab6994, Abcam). To visualize activated microglia, anti-rat CD11b (Integrin alpha M) antibody (ab133357, Abcam) was used. Reactive astrocytes were visualized by staining with anti-rat GFAP (glial fibrillary acidic protein) antibody (ab53554, Abcam). Immunofluorescence PSMA staining was applied to tumor bearing brain slices using three different anti-PSMA antibodies (NBP1-45057 and NBP1-89822, Novus Biologicals; ab58779, Abcam) to display PSMA expression. To verify the functionality and specificity of the used PSMA antibodies, rat prostate and kidney tissue were used as positive controls. Immunofluorescence stainings were performed according to standard histology protocols and as described before [19 (link)].
Blood vessels were stained with anti-rat von Willebrand factor antibody (ab6994, Abcam). To visualize activated microglia, anti-rat CD11b (Integrin alpha M) antibody (ab133357, Abcam) was used. Reactive astrocytes were visualized by staining with anti-rat GFAP (glial fibrillary acidic protein) antibody (ab53554, Abcam). Immunofluorescence PSMA staining was applied to tumor bearing brain slices using three different anti-PSMA antibodies (NBP1-45057 and NBP1-89822, Novus Biologicals; ab58779, Abcam) to display PSMA expression. To verify the functionality and specificity of the used PSMA antibodies, rat prostate and kidney tissue were used as positive controls. Immunofluorescence stainings were performed according to standard histology protocols and as described before [19 (link)].
4
Profiling Gene Expression in Tumor Subtypes
5
Laser Microdissection and DNA Extraction from FFPE Tissue
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FFPE tissue sections (10 µm) were prepared using a DIRECTOR slide (cat. no. 11505158; Leica Microsystems GmbH). Following deparaffinization, the sections were subjected to LMD using an LMD 6000 (Leica Microsystems) according to the protocol provided by the manufacturer. DNA was extracted from FFPE samples using a RecoverAll Total Nucleic Acid Isolation kit (Thermo Fisher Scientific, Inc.) with certain modifications, as described previously (27 (link)). DNA quality was determined by performing quantitative PCR (qPCR) for GAPDH using a TaqMan® Gene Expression Assay kit (cat. no. Hs02758991_g1; Thermo Fisher Scientific, Inc.). As a quality check control, the ratio of sample DNA to frozen tissue DNA was calculated using the delta quantification cycle (ΔCq) method and the results are expressed as 2−ΔΔCq, as described previously (27 (link)–29 (link)).
6
Laser Microdissection and DNA Extraction from Tumor Samples
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Tumour and normal gland (reference) samples were obtained from 5-micron-thick tissue sections using a laser microdissection system (LMD6000; Leica Microsystems, Wetzlar, Germany). Each sample was dissected from an area of ≥6 mm2. In tumour samples, neoplastic cells comprised 90% of the total cell count. These cells were then digested in a 200-mg/mL proteinase K (P2308, Sigma-Aldrich, St. Louis, USA) solution for 70 ± 2 h at 37°C prior to the phenol/chloroform DNA extraction. Corresponding tumour areas were assessed for the immunohistochemical staining of mucin and other markers as described above. DNA quality was assessed based on the A260/A280 ratio (cut-off. >1.5) and A260/A230 ratio (cut-off. > 1.0) and by the presence or absence of double-stranded DNA.
7
Laser Microdissection for Genomic DNA
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All the samples stored at −80 °C were firstly made into frozen section, and HCC and liver cirrhosis tissues with typical histopathological characteristics were observed and selected, respectively. Then eight serial 10-μm tissue sections were prepared and placed on UV-absorbing membrane for laser microdissection by LMD6000 (Leica Microsystems Ltd, Wetzlar, Germany). After HE-staining, the slides were mounted on microstat, and the selected HCC lesion and their adjacent DN were dissected by a UV laser in mode of motorized optical beam scanning, respectively. The dissectate (with the attached specimen) dropped by its gravity into the cap of a 0.5-mL microcentrifuge tube that was filled with 40 μL lysate buffer and 10 μL protease K. Along with each dissected HCC and DN, surrounding normal liver tissue of the same size was isolated and analyzed as a control. The microcentrifuge tubes were placed in a waterbath (48 °C) to digest the tissue specimens, and then extract DNA using QiaGen kit (Germany). Finally, genomic DNA was confirmed by gel electrophoresis (20 g/L agarose) and stored at −20 °C until using.
8
Microarray Analysis of Laser-Microdissected Cancer Cells
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Cancer cells were isolated from sectioned tissues by laser microdissection using the LMD6000 (Leica microsystems, Wetzlar, Germany). Total RNA was extracted using the RNeasy Micro Kit (QIAGEN, Hilden, Germany) and was amplified, biotinylated, and fragmented using the MessageAmp II-Biotin Enhanced Kit (Ambion, Austin, TX, USA) according to the manufacturers' instructions. The fragmented aRNA was applied to GeneChip Human Genome U133 Plus 2.0 (Affymetrix, Santa Clara, CA, USA) and incubated at 45 °C for 16 h, followed by washing and staining using Fluidic Station 450 (Affymetrix). The hybridized probe arrays were scanned with the GeneChip Scanner 3000 7G (Affymetrix). Microarray data used in this study are available at the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/ ) under the accession number GSE52915.
9
Spatial Transcriptomic Analysis of Rat Brain
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Frozen sections of rat brains (13 μm thick) at each stage of development (E20, P0, P15, and 6–8 weeks adult) were cut on Cryostat CM1950 (Leica Microsystems) and mounted on PEN-membrane coated glass slides (Leica Microsystems). After fixation in 70% ethanol and staining with hematoxylin, 1000 cells from meninges and 6–8 weeks adult SVZ were dissected with LMD6000 instrument (Leica Microsystems). Cells were collected in the cap of 0.5 ml tube containing the lysis buffer from Picopure RNA Isolation kit (Arcturus) and RNA extraction was performed according to manufacturer's protocol. First strand cDNA was synthesized with random primers using SuperScript II Reverse Transcriptase (Invitrogen) and used for subsequent qRT-PCR analysis.
10
Laser Microdissection of Condensed and Scattered Cell Areas
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To separate CS condensed and scattered areas, laser microdissection was used with the Leica laser microdissection system LMD 6000 (Leica Microsystems, Wetzlar, Germany). The general principle was described in Reference [35 (link)]. Briefly, MSC were seeded on microdissection dishes with a polyethylene bottom (FWST-5030, WillCo Wells B.V., Amsterdam, The Netherlands). Obtained as previously described, the CS were fixed with 70% ethanol, and the condensed and scattered areas were microdissected under visual control. The dissected areas were collected with a sterile 1.5-mL polypropylene tube (Eppendorf, Hamburg, Germany). The obtained samples (n ≈ 50 per tube) were lysed in appropriate buffer and used for downstream applications: Western blotting, solid-phase dot-ELISA, PicoGreen DNA assay, LDH activity assay and RNA isolation for RNA-sequencing.
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