Laser capture microdissection (LCM) was performed to specifically isolate tissue of three distinct structures: 1. Myocardial continuity sinus venosus and AV canal (SV-AVC); 2. More caudal region of posterior AV canal (AVC); 3 Lateral wall right ventricle (RV). Embryos of HH21 were extracted and fixated for 24 hrs in 4% paraformaldehyde, embedded in paraffin and serially sectioned at 5 μm. Sections were mounted on membrane-covered slides (MembraneSlide 1.0 PEN; Carl Zeiss Microscopy, Thornwood, NY, USA). Tissue from the different structures of interest were dissected using the PALM microbeam (Carl Zeiss Microscopy) and RoboSoftware package and subsequently transferred to AdhesiveCaps (Carl Zeiss Microscopy). After microdissection, the tissue was stored at −80°C. The outline of the dissected tissue was photographed in all embryos and examples of the different regions before and after dissection are shown in Figure S1 .
Adhesive caps
Manufactured by Zeiss
Sourced in Germany
Adhesive caps are a type of laboratory equipment designed to seal the tops of various containers, such as test tubes or vials. Their primary function is to provide a secure and reliable closure to prevent the contents from spilling or evaporating during handling, storage, or transportation.
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Lab products found in correlation
Palm microbeam, by Zeiss (3 mentions)
1.0 pen membrane slides, by Zeiss (2 mentions)
Palm microbeam laser microdissection system, by Zeiss (2 mentions)
Leica lmd6500 system, by Leica Microsystems (2 mentions)
Absolutely rna nanoprep kit, by Agilent Technologies (2 mentions)
Smarter stranded total rna seq kit v2 pico input mammalian, by Takara Bio (2 mentions)
Palm lcm system, by Zeiss (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Picopure rna isolation kit, by Arcturus (2 mentions)
Membraneslide 1.0 pen, by Zeiss (1 mentions)
Leica rm2235, by Leica Biosystems (1 mentions)
Membrane slide, by Zeiss (1 mentions)
Cryostat, by Leica camera (1 mentions)
Qiazol, by Qiagen (1 mentions)
Polyethylene naphthalate membrane slides, by Zeiss (1 mentions)
Palm microbeam lcm system, by Zeiss (1 mentions)
Palm microbeam 4, by Zeiss (1 mentions)
Cm1950 cryotome, by Leica camera (1 mentions)
Cresyl violet acetate, by Merck Group (1 mentions)
Axio observer, by Zeiss (1 mentions)
19 protocols using adhesive caps
1
Laser Capture Microdissection of Cardiac Structures
2
Laser Microdissection of Cancer and Adjacent Normal Tissue from FFPE Samples
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FFPE tissues acquired from surgical excisions or biopsies were collected for analysis. Before performing LMD, pathologists classified the cancer and ANT areas. ANTs were defined as epithelial tissues located on the surgical margin that were ≥5 mm away from cancer tissues. A microtome (Leica RM2235; Leica Biosystems Nussloch GmbH, Nussloch, Germany) was used to prepare 7-µm-thick tissue sections from FFPE tissue blocks, which were placed on nuclease-free 1.0 PEN Membrane Slides (no. 415190-9081-000; Zeiss GmbH, Jena, Germany). Then, tissue sections were deparaffinized, stained with cresyl violet, and dried in air briefly, and the slides were subsequently stored at −20°C. LMD was performed using a Zeiss PALM Microbeam laser microdissection system (Carl Zeiss Microscopy, Jena, Germany) and PALM Robo v4.6 software. For capturing tissue, Adhesive Caps (no. 415190-9201-000; Zeiss GmbH) were used. Tissue sections (cancer tissue and paired ANT) were immediately deparaffinized and total RNA was then extracted.
3
Laser Capture Microdissection of Cortical Layers
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Twenty µm thick frozen tissue sections were mounted on glass slides coated with polyethylene naphthalate, fixed in 100% ethanol for 20 min, and subsequently air-dried and rehydrated twice in sterile H2O for 1 min. Next, tissue sections were incubated in toluidine (1% w/v in sterile H2O) for 1 min at room temperature and washed in sterile H2O 3 times for 1 min. Sections were then dehydrated in 100% ethanol twice for 3 min and air-dried. Laser capture microdissection was performed using a Leica LMD6500 system (Leica Microsystems, Wetzlar, Germany). All cortical layers from the middle frontal gyrus were included. Microdissected tissue samples of 100 mm3 were collected into adhesive caps (Zeiss, Göttingen, Germany) and stored at −80 °C until use.
4
Laser Microdissection of Tumor Cells
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Prior to microdissection, 10-µm sections were cut from the frozen tissue samples using a cryostat (Leica, Wetzlar, Germany) and stained with hematoxylin and eosin by standard methods. These sections were used to ensure the presence of tumor tissue in the samples and to define dissection margins (Figure 5 ).
For laser-microdissection, 20-µm sections of tissue samples were mounted on Zeiss membrane slides (Carl Zeiss microimaging, Jena, Germany) and stained with cresyl violet using an laser capture microdissection Staining Kit (Ambion®/Applied Biosystems, Darmstadt, Germany). Subsequently, laser-microdissection was conducted using a PALM® MicroBeam Laser System (PALM® Microlaser Technologies AG, Bernried, Germany) to separate tumor cells from stromal cells. Tissue from the respective host organs (liver and lung parenchyma) was collected at least 3 cm away from the tumor bulk. Microdissected tissue was transferred to adhesive caps (Carl Zeiss, Jena, Germany), lysed in QIAzol® (Qiagen, Hilden, Germany), and stored at −80 °C until final evaluation.
For laser-microdissection, 20-µm sections of tissue samples were mounted on Zeiss membrane slides (Carl Zeiss microimaging, Jena, Germany) and stained with cresyl violet using an laser capture microdissection Staining Kit (Ambion®/Applied Biosystems, Darmstadt, Germany). Subsequently, laser-microdissection was conducted using a PALM® MicroBeam Laser System (PALM® Microlaser Technologies AG, Bernried, Germany) to separate tumor cells from stromal cells. Tissue from the respective host organs (liver and lung parenchyma) was collected at least 3 cm away from the tumor bulk. Microdissected tissue was transferred to adhesive caps (Carl Zeiss, Jena, Germany), lysed in QIAzol® (Qiagen, Hilden, Germany), and stored at −80 °C until final evaluation.
5
Adrenal Cortex Microdissection Protocol
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Adrenal glands frozen in liquid nitrogen were cut in 25–30 μm thick sections, mounted on polyethylene naphthalate membrane slides (Zeiss), dehydrated in increasing concentrations of ice-cold ethanol (75%, 95%, 100%) for 45 s each, and air-dried at room temperature (RT). Laser capture microdissection was performed with a Zeiss PALM MicroBeam LCM system. The adrenal cortex from 8 to 12 sections was microdissected and the tissue was collected on Adhesive Caps (Zeiss).
6
Laser Capture Microdissection of Frozen Tumor Tissue
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Frozen tumour tissue was fixed for 10 min with 100% ethanol and air dried. Following fixation 200–400 HRS or surrounding bystander cells were excised using a Zeiss PALM LCM system and catapulted onto adhesive caps (415190-9191-000—Zeiss). Cell lysis, RNA extraction and purification were performed using the absolutely RNA Nanoprep Kit (400753—Agilent). RNA-Seq libraries were produced using the SMARTer Stranded Total RNA-Seq Kit v2—Pico input Mammalian (Takara Bio). The libraries were used for both rtPCR as previously described and were sequenced on an Illumina NextSeq 500 at the Genomics Birmingham facility.
7
Laser Capture Microdissection of Infected Tissue
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The FFPE sections were fixed onto 1.0 PEN membrane slides (Zeiss, Jena, Germany) and stained with Mayer’s hematoxylin to aid in visualization, as described previously [31 (link)]. Regions of interest containing bacteria or regions free from infection were identified and targeted for dissections by the Arcturus laser capture microdissection system (Life Technologies). Dissected tissue was captured on Adhesive Caps (Zeiss) for further processing. One spot from the region of interest was cut from five adjacent tissue sections and pooled for analysis. The micro-dissected tissue was fragmented with trypsin, using a LiquidTissue digestion kit (Expression Pathology, Rockville, MD) according to the manufacturer’s directions.
8
Laser Capture Microdissection of Kidney Samples
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Tissue sections (8 µm) were cut from FFPE blocks onto Zeiss Membrane Slides (1.0 PEN, cat. no. 415190‐9041‐000, Carl Zeiss Microscopy, Thornwood, NY, USA). The Ambion LCM Staining Kit (Life Sciences, Grand Island, NY, USA) was employed according to the manufacturer's instructions, with modifications as reported by Cianciolo et al.21 . Tissue sections were cleared by incubations in xylene, then briefly air dried before LCM. The slides were stored at 4 °C when not on the LCM stage.
All LCM was performed using a Zeiss PALM Microbeam laser microdissection system (Carl Zeiss Microscopy, Thornwood, NY) with PALMRobo v4.6 software. Tissue was captured into AdhesiveCaps (#415190‐9201‐000, Carl Zeiss Microscopy, Thornwood, NY), and DNA isolated from captured tissue using the Ambion RecoverAll system as described below.
Approximately 100 cross‐sections from normal kidney tubules, distant from the neoplasm, were collected by LCM. Each cross‐section contained, on average, approximately 18 (range 6–37) nuclei for a total recovery of approximately 1800 nuclear equivalents/microdissected sample. Approximately equivalent numbers of tumour nuclei were also microdissected from areas of both invasive and in situ urothelial carcinoma.
All LCM was performed using a Zeiss PALM Microbeam laser microdissection system (Carl Zeiss Microscopy, Thornwood, NY) with PALMRobo v4.6 software. Tissue was captured into AdhesiveCaps (#415190‐9201‐000, Carl Zeiss Microscopy, Thornwood, NY), and DNA isolated from captured tissue using the Ambion RecoverAll system as described below.
Approximately 100 cross‐sections from normal kidney tubules, distant from the neoplasm, were collected by LCM. Each cross‐section contained, on average, approximately 18 (range 6–37) nuclei for a total recovery of approximately 1800 nuclear equivalents/microdissected sample. Approximately equivalent numbers of tumour nuclei were also microdissected from areas of both invasive and in situ urothelial carcinoma.
9
FFPE Lung Tumor LCM and AMPK Analysis
10
Cryosectioning and Laser Capture Microdissection of Endometrial Epithelium
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Endometrial cryosections of the in vivo– and field sampling–obtained samples were prepared for each tissue piece with a Leica CM 1950 Cryotome. The tissue was embedded in optimal cutting temperature compound (Biosystems, category no. 3801480S), and 10-µm sections were cut at a temperature of −25 °C and −20 °C for the cryo-chamber and specimen head. Sections were mounted on 1.0 PEN NF Membrane Slides (Zeiss, cat. no. 415190–9081-000) and stained with 1% (wt/vol) Cresyl violet acetate (Sigma Aldrich, cat. no. 86098–0) in 50% EtOH. The stained sections were stored at −80 °C until further processing. The intercaruncular luminal epithelium was collected with the Carl Zeiss Axio Observer with the AxioCam Cc1 camera, a LD Plan-NEOFLUAR 20× objective, the PALM MicroBeam laser, and the PALMRobo software (release 4.8, version 4.8.0.1). Samples were cut with an energy of 46 and a focus of 64. The laser pressure catapulting had an energy delta of 20 and a focus delta of 2. LE cells were collected in Adhesive Caps (Zeiss, cat. no. 415190–9191-000). All samples were stored at −80 °C until nucleic extraction.
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