Frozen sections were defrosted stained within 5 min using H & E staining. In short, sections were hydrated in a series of 100%, 95% and 80% alcohol and H20, followed by Hematoxilyin, Acid Alcohol (1%) and Eosin. Sections were then dehydrated in an ethanol series (95%, 100% ethanol, and 100% xylene) and dried. All aqueous steps and stains contained a 1X ProtectRNA™ RNase Inhibitor (Sigma-Aldrich, St. Louis, MO). Immediately after staining, the DG region and PC from CA1–CA3 were captured in ADhesiveCap tubes (Carl Zeiss, Oberkochen, Germany) with a PALM microbeam LCM (Axiovert 200M, Carl Zeiss, Oberkochen, Germany). For samples which more than 300 cells were isolated, RNA was extracted using the PicoPure RNA Isolation Kit (Life Technologies, Carlsbad, CA). For the entire HP the section was scraped from the slide and RNA was isolated using TRIzol and purified using the PureLink RNA Mini Kit (Life Technologies, Carlsbad, CA), but not LCM was performed. The same RNA isolation and purification methods were also used for the EC. Finally, the RNA extracted from all regions was quantified using the Quant-iT™ RiboGreen® RNA Assay Kit (Thermo Fisher Scientific, MA).
Adhesivecap tube
Manufactured by Zeiss
Sourced in Germany
AdhesiveCap tubes are laboratory equipment designed for sample collection and storage. The tubes feature an adhesive cap that securely seals the sample, ensuring sample integrity during transport and storage.
Automatically generated - may contain errors
Lab products found in correlation
Palm microbeam, by Zeiss (4 mentions)
Thermoscript reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
P a l m laser microdissection system, by Zeiss (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Rneasy ffpe kit, by Qiagen (2 mentions)
Realplex4 cycler, by Eppendorf (2 mentions)
Axiovert 200m, by Zeiss (1 mentions)
Quant it ribogreen rna assay kit, by Thermo Fisher Scientific (1 mentions)
Picopure rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Protectrna rnase inhibitor, by Merck Group (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Lmd7000, by Leica (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Pen membrane slide, by Leica (1 mentions)
Lmd7000 system, by Leica (1 mentions)
Zero blunt topo vector, by Thermo Fisher Scientific (1 mentions)
Membrane slide, by Zeiss (1 mentions)
Palm microbeam system, by Zeiss (1 mentions)
Qiaamp dna ffpe tissue kit, by Qiagen (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
11 protocols using adhesivecap tube
1
Laser Capture Microdissection of Hippocampal Regions
2
Laser Microdissection of Mouse Kidney Structures
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Nine-micrometer thick cryosections from snap-frozen mouse kidneys (n = 5/group) were fixed, stained, and after washing and air-drying, sections were used for laser microdissection (LMD, Leica LMD7000; Leica Microsystems, Germany) to collect arteriolar vascular segments (minimum area 5 × 105 μm2), glomeruli (minimum area 1 × 106 μm2), and venules (minimum area 1 × 106 μm2). Laser microdissected samples were collected in 0.5 mL AdhesiveCap tubes (ZEISS, Göttingen, Germany) and stored at −80°C for further analysis. Leftover cryosections from LMD was collected in 350 μL RLT buffer (Qiagen) as post-LMD samples and stored at −80°C until further analysis.
3
Laser Capture Microdissection of PDAC Tumors
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LCM was performed on the PDX as previously described [11 (link)]. Briefly, cresyl violet stained slides were brought to room temperature. Tumour cells from each cresyl violet section were microdissected using the PALM LMPC device (Carl Zeiss MicroImaging, GmbH, Munich, Germany). Tissue was collected in AdhesiveCap tubes (Carl Zeiss MicroImaging, GmbH, Munich, Germany) and stored at -80°C prior to extraction.
LCM of fresh frozen tissue samples from PDAC was performed on a Leica LMD 7000 instrument. Frozen tissue for tumour samples was maintained in vapor-phase liquid nitrogen and embedded in OCT cutting medium and sectioned in a cryotome into 8-μm thick sections. These sections were then mounted on PEN membrane slides (Leica) and lightly stained with hematoxylin to distinguish tumour epithelium from stroma. A pathologist (SF) marked tumour sections and LCM was performed on the same day according to manufacturer’s protocol on the Leica LMD7000 system. Microdissected tumour cells were collected by gravity into the caps of sterile, RNAse-free microcentrifuge tubes. Approximately 150,000–200,000 tumour cells were collected for each DNA extraction and stored at –80°C in Arcturus PicoPure Extraction Buffer.
LCM of fresh frozen tissue samples from PDAC was performed on a Leica LMD 7000 instrument. Frozen tissue for tumour samples was maintained in vapor-phase liquid nitrogen and embedded in OCT cutting medium and sectioned in a cryotome into 8-μm thick sections. These sections were then mounted on PEN membrane slides (Leica) and lightly stained with hematoxylin to distinguish tumour epithelium from stroma. A pathologist (SF) marked tumour sections and LCM was performed on the same day according to manufacturer’s protocol on the Leica LMD7000 system. Microdissected tumour cells were collected by gravity into the caps of sterile, RNAse-free microcentrifuge tubes. Approximately 150,000–200,000 tumour cells were collected for each DNA extraction and stored at –80°C in Arcturus PicoPure Extraction Buffer.
4
EGFP-labeled Neuron Transcriptome Analysis
5
Laser Capture Microdissection of Diseased Transgenic Brains
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LCM was performed using a Zeiss PALM MicroBeam system. Diseased transgenic brains were processed as for pathology and then mounted on special MembraneSlide (Zeiss) slides at a thickness of 7 μm. Before LCM capturing, the slides were de-paraffinised using HistoClear II solution (VWR) twice for 5 minutes each. The slides were allowed to dry completely after dehydration in 100% ethanol twice for 10 minutes each. Different areas of interest were captured in special AdhesiveCap tubes (Zeiss). Extraction of genomic DNA was carried with using a QIAamp DNA FFPE Tissue Kit (Qiagen) according to manufacturer’s instructions. For PCR of the recombined (AG2 band) and non-recombined (GFP band) transgene, a previously described method was used1 (link). High fidelity amplified bands (Q5 polymerase, NEB) were cut from an agarose gel and purified using Qiagen MinElute kit and cloned into Zero-Blunt TOPO vector (Life Technologies) before transformation into chemically competent DH5α E.coli. Three individual clones were selected from Kanamycin plates for each of the AG2 and GFP bands and sequenced using SP6, M13 or T7 primers in the forward and reverse directions. Relevant sequences are shown in Supplementary Fig. 1 . Alignment of the sequenced bands with the reference sequence was done using BLAST at NIH.
6
Retrograde Labeling of Dopaminergic Neurons
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For retrograde labeling of DA neurons, mice were anesthetized with ketamine/xylazin, placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA), and received instrastriatal (bregma +0.4 mm; lateral +/–1.8 mm; ventral –3.5 mm) injections of 300 nl red fluorescent retrobeads (Lumafluor, Naples, FL, USA). Animals were killed after 1–2 weeks, the brains dissected and used for cryosectioning. Serial coronal sections (7 μm) of the midbrain spanning the region between bregma –3.4 to –3.6 were mounted on nuclease-free membrane slides (Zeiss, Oberkochen, Germany), fixed in ascending ethanol series and used for laser-assisted microdissection of fluorescently labeled DA neurons in the SN from both sides on the brain as described in.4 (link) Individual neurons (approximately 2000 per mouse) were dissected on a Zeiss PALM MicroBeam system (Zeiss) and catapulted into AdhesiveCap tubes (Zeiss). Total RNA including miRs was isolated from the dissected samples with a miRNeasy Micro kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions.
7
Laser Microdissection of Mouse PVN
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Mouse brains from 9-week-old, 129/Sv Mrap2tm1a/tm1a and Mrap2+/+ mice were dissected, immediately embedded into OCT and frozen in liquid nitrogen. Coronal sections (20μm) covering the region from −0.58mm to −1.22mm caudal to bregma (Franklin & Paxinos 2012 ) were cut on a cryostat and mounted on Superfrost Plus slides (Thermo-Fisher). Frozen sections were fixed for 40s in 95% ethanol and then rehydrated (75% and 50% ethanol, 30s each). The slides were stained with 1% cresyl violet in 75% ethanol (w/v) for 45s, dehydrated in a graded ethanol series (50, 75, 95, 100% for 30s each), in 100% ethanol for 5min and air-dried. Laser microdissection was performed using a P.A.L.M. MicroBeam (Zeiss). The PVN was collected into AdhesiveCap tubes (Zeiss). Total RNA was immediately isolated using the RNAqueous-Micro Kit (Ambion). Quality and quantity of the total RNA samples were determined by the Agilent BioAnalyzer using PicoChip. RNAse free technique and RNAase free reagents were used throughout.
8
EGFP-labeled Neuron Transcriptome Analysis
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35 μm sections of labeled brains were stained for EGFP, and mounted on membrane PEN slides (Carl Zeiss). Labeled neurons were visualized, and captured into adhesive cap tubes (Carl Zeiss) using a PALM laser microdissection system (Carl Zeiss) using a uv laser to cut and flip tissue samples directly into inverted adhesive caps placed above slides. RNA was extracted using an RNeasy FFPE kit (QIAGEN). Reverse transcription was performed using random hexamers and Thermoscript reverse transcriptase (Fisher Scientific). Real time PCR was performed using SYBR® Green PCR Master Mix (Thermo Fisher) on a Realplex4 cycler (Eppendorf). We confirmed the specificity and size of the amplicons by running the PCR product on agarose gels, and by melting curve analysis. For Klhl14-overexpression experiments, data were analyzed using an unpaired two-sided t test. The PCR primers are listed in Key Resources Table .
9
Laser Capture Microdissection of Oogenesis Follicle Cells
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LCM was performed with a Zeiss PALM MicroBeam and visualized under a 63X objective. Sectioned mid-oogenesis egg chambers were staged according to morphological criteria. Once stage 9–10 egg chambers had been identified, either the apical half (“apical fragment”) or the basal half (“basal fragment”) of 5-10 contiguous columnar follicle cells was microdissected and collected into the cap of an AdhesiveCap tube (Zeiss). 10 fragments of either apical or basal sample type from different egg chambers were pooled for each replicate, with a total microdissected area of ~3000–4000 μm2/replicate. LCM samples were processed according to Chen et al.88 (link) to produce high-quality Illumina sequencing libraries. Samples were multiplexed and simultaneously sequenced in a single lane using the NextSeq500 system according to the manufacturer’s instructions.
10
Laser Capture Microdissection of Skin Epidermal Cells
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Human skin sections (10-μm thickness) were placed on PEN (polyethylene naphthalate) membrane–covered slides (415190-9041-000, Carl Zeiss Microscopy), which were heated at 180°C for 4 hours and irradiated with ultraviolet (UV) light for 30 min before their use. Sections were dried at −20°C, fixed with 70% ethanol for 2 min, and stained with cresyl violet acetate solution (C5042, Sigma-Aldrich). Slides were shortly air-dried after exposure to an increasing gradient of ethanol solutions. Sections were microscopically analyzed using the Axio Observer Z1 (Carl Zeiss Microscopy). Laser capture microdissection was conducted using a UV laser of the PALM MicroBeam (Carl Zeiss Microscopy) and the PALM RoboSoftware (Carl Zeiss Microscopy). Epidermal areas of the skin sections were therefore labeled, and the RoboLPC function was used to microdissect and catapult the tissue into the cap of a 0.2-ml AdhesiveCap tube (Carl Zeiss Microscopy). Laser capture microdissected epidermal areas from three serial sections of one individual donor or patient were collected for further RNA extraction.
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