Frozen sections mounted onto membrane-slides (Carl Zeiss Microscopy) were fixed in 70% ethanol, and washed with RNase-free distilled water for 5 min. Sections were stained with Mayer’s hematoxyline, and then rinsed with RNase-free water. After that, dehydration was performed in a quick increasing ethanol series. LCM was performed using a PALM Micro Beam System (Zeiss Instruments). Samples from each group were collected and pooled per group into an Adhesive Cap 500 opaque (Carl Zeiss Microscopy) and stored in QIAzol Lysis Reagent (QIAGEN) at −80°C. RNA extraction was performed using the miRNeasy Micro Kit (QIAGEN). Complementary DNA was synthesized using iScript cDNA Synthesis Kit (Bio Rad). Quantitative real time RT-PCR was performed in triplicate using TaqMan Universal PCR Master Mix (Life Technologies) on a StepOnePlus Real-Time PCR System (Life Technologies). Fluorogenic probes and primers were used to detect: IL-6 (Mm00446190_m1), MIP-2 (Mm00436450_m1), and TLR2 (Mm00442346_m1) for mouse samples (Life Technologies). Expression of target gene was normalized to β-actin. Expression was analyzed by the 2−ΔΔCt method.
Adhesivecap 500 opaque
Manufactured by Zeiss
Sourced in Germany
The AdhesiveCap 500 opaque is a laboratory equipment product manufactured by Zeiss. It is designed to provide a secure and reliable sealing solution for various types of sample containers or microplates. The product features an opaque cap that helps to protect the contents from external light and environment.
Automatically generated - may contain errors
Lab products found in correlation
Palm microbeam, by Zeiss (6 mentions)
Rneasy micro kit, by Qiagen (5 mentions)
Isogen, by Nippon Gene (4 mentions)
T100 thermal cycler, by Bio-Rad (4 mentions)
Palm microbeam system, by Zeiss (2 mentions)
Membrane slide, by Zeiss (1 mentions)
Il 6 mm00446190 m1, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Dntp mixture, by Takara Bio (1 mentions)
Rneasy ffpe kit, by Qiagen (1 mentions)
Axiovert 200 inverted microscope, by Zeiss (1 mentions)
Ambion single cell to ct kit, by Thermo Fisher Scientific (1 mentions)
Maxima h minus cdna synthesis master mix, by Thermo Fisher Scientific (1 mentions)
T100, by Bio-Rad (1 mentions)
9 protocols using adhesivecap 500 opaque
1
Laser Capture Microdissection and qRT-PCR Analysis
2
Laser Microdissection and RNA Extraction from Skin Tissue
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LMD was performed using a PALM MicroBeam (Carl Zeiss, Oberkochen, Germany). Manufacturer-recommended slide and collection tubes (AdhesiveCap 500 opaque, Carl Zeiss) were set up, and after adjusting the aperture and intensity using a 20× magnification objective lens, only the epidermis and dermis structures of the wound were carefully cut from the tissue. Tube caps were filled with Buffer RLT (RNeasy Micro Kit, Qiagen, Hilden, Germany) with β-mercaptoethanol to allow isolation of intact RNA. Total RNA was extracted from cells or skin tissues using a monophasic solution of phenol and guanidine isothiocyanate (ISOGEN; NipponGene, Tokyo, Japan) according to the manufacturer’s instructions. Total RNA was mixed with random primers, reverse transcriptase, and dNTP mixture (TakaraBio) and incubated in a T100TM thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 25 °C for 5 min, 55 °C for 10 min, and 80 °C for 10 min to produce cDNA.
3
Laser-Assisted Endothelial Cell Isolation
4
Laser Microdissection and RNA Extraction
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LMD was performed using PALM MicroBeam (Carl Zeiss, Germany). The manufacturer’s recommended slides and collection tubes (AdhesiveCap 500 opaque; Carl Zeiss) were set up, and the tissue was carefully cut after adjusting the aperture and intensity using a 20× magnification objective lens. The tube caps were filled with Buffer RLT (RNeasy Micro Kit; Qiagen, Germany) with β-mercaptoethanol to allow the separation of intact RNA. Total RNA was extracted from cells or skin tissue using a monophasic solution of phenol and guanidine isothiocyanate (ISOGEN; Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. Total RNA was mixed with a random primer, reverse transcriptase, and dNTP mixture (Takara Bio). Reverse transcription was performed on a T100TM thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 25 °C for 5 min, 55 °C for 10 min, and 80 °C for 10 min to heat-inactivate the reverse transcriptase and produce cDNA.
5
Laser Capture Microdissection of Breast Cancer
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Twenty-four formalin-fixed, paraffin-embedded (FFPE) breast cancer specimens were selected (twelve basal-like, six luminal A and six luminal B). All basal-like samples were also triple negative, and all luminal samples were ER and PR positive, and HER2 negative. The luminal B tumours displayed more than 15% Ki67-positive nuclei (whole section). All were diagnosed as invasive carcinomas (NST; previously ductal carcinomas). Ten μm thick FFPE sections were deparaffinised, rehydrated and stained with haematoxylin. Breast cancer epithelium was laser micro-dissected (PALM MicroBeam, Zeiss) and pressure catapulted into a tube cap (AdhesiveCap 500 opaque, Zeiss). Depending on the available tissue, 0.8–1.9 × 107 µm3 were micro-dissected.
6
Laser Capture Microdissection of Fluorescent Cells
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We used the PALM MicroBeam System (Zeiss) equipped with a fluorescence microscope. Each fluorescently labeled cell was selected under the microscope with an LD Plan-Neofluar objective lens (40×/0.6 numerical aperture), and laser captured into an AdhesiveCap 500 opaque (Zeiss). A sample of up to ∼100 cells were collected in a single AdhesiveCap and lysed with cell lysis buffer. When the collection took more than 1 h, we lysed cells to minimize sample degradation and continued collection in a new AdhesiveCap.
7
LMD and Total RNA Extraction from Cells and Tissues
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LMD was performed using a PALM MicroBeam (Carl Zeiss, Oberkochen, Germany). The manufacturer’s recommended slides and collection tubes (AdhesiveCap 500 opaque, Carl Zeiss) were set up, and the tissue was carefully cut after adjusting the aperture and intensity using a 20× magnification objective lens. The tube caps were filled with Buffer RLT (RNeasy Micro Kit; Qiagen, Hilden, Germany) and β-mercaptoethanol to allow the separation of intact RNA. Total RNA was extracted from cells or skin tissues using a monophasic solution of phenol and guanidine isothiocyanate (ISOGEN; NipponGene, Tokyo, Japan) according to the manufacturer’s instructions. Total RNA was mixed with a random primer, reverse transcriptase, and dNTP mixture (Takara Bio, Tokyo, Japan). The mixture was incubated in a T100TM thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) at 25 °C for 5 min, 55 °C for 10 min, and 80 °C for 10 min to heat inactivate the reverse transcriptase and synthesize cDNA.
8
LMD-Based RNA Extraction and cDNA Synthesis
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LMD was performed using a PALM MicroBeam (Carl Zeiss, Oberkochen, Germany). The manufacturer’s recommended slides and collection tubes (AdhesiveCap 500 opaque, Carl Zeiss) were set up, and the tissue was carefully cut after adjusting the aperture and intensity using a 20× magnification objective lens. The tube caps were filled with Buffer RLT (RNeasy Micro Kit; Qiagen, Hilden, Germany) and β-mercaptoethanol to allow separation of intact RNA. Total RNA was extracted from cells or skin tissues using a monophasic solution of phenol and guanidine isothiocyanate (ISOGEN; NipponGene, Tokyo, Japan) according to the manufacturer’s instructions. Total RNA was mixed with a random primer, reverse transcriptase, and dNTP mixture (Takara Bio, Tokyo, Japan). The mixture was then incubated in a T100TM thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) at 25 °C for 5 min, 55 °C for 10 min, and 80 °C for 10 min to heat inactivate the reverse transcriptase and synthesize cDNA.
9
Laser Microdissection for RNA Extraction
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We performed LMD using a PALM MicroBeam (Carl Zeiss, Oberkochen, Germany) to collect RNA from the tissue. Briefly, the manufacturer’s recommended slides and collection tubes (AdhesiveCap 500 opaque, Carl Zeiss) were set up, and the samples were collected from the wound margins by carefully cutting the tissue while observing with a 20× magnification objective lens. The tube cap was filled with Buffer RLT (RNeasy microkit, Qiagen, Hilden, Germany) and filled with β-mercaptoethanol to allow the separation of intact RNA using the RNeasy microkit (Qiagen). Total RNA was extracted from the cells or skin tissues according to the manufacturer’s instructions, and then placed in a T100TM thermal cycler (Bio-Rad, Hercules, CA, USA) along with Maxima™ H Minus cDNA Synthesis Master Mix (ThermoFisher Scientific, Waltham, MA, USA) at 25 °C for 5 min, 55 °C for 10 min, and 80 °C for 10 min to thermally inactivate revertase and produce cDNA.
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