The brains were extracted, fixed in 4% paraformaldehyde, dissected at the brainstem level using a mouse brain slicer matrix (RBMA–200C, World Precision Instruments), paraffin embedded, cut in 2 μm thick sections, and scanned using an Olympus BX51 microscope coupled with a DotSlide system for the identification of RVLM. TH staining (dilution 1:50, Cat. Number 22941, ImmunoStar) was carried out using the Ventana Discovery system (Roche) and the following parameters: citrate buffer pH 6.0 for heat–induced antigen retrieval, 40 min incubations with primary and secondary (Cat. number ab133469, Abcam) antibodies, detection step with the ChromoMap DAB kit (Cat. number 760–159, Roche), and counterstaining with hematoxylin. The quantification of the number of RVLM TH+ neurons was done using standard microscopy analyses.
Rbma 200c
Manufactured by World Precision Instruments
The RBMA-200C is a precision laboratory balance designed for weighing samples. It features a range of 0.1 mg to 200 g, with a readability of 0.1 mg. The balance has a stainless steel weighing platform and a durable body construction.
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Lab products found in correlation
Axio zoom v16, by Zeiss (2 mentions)
Ventana discovery system, by Roche (1 mentions)
Chromomap dab kit, by Roche (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Truseq stranded rna kit, by Illumina (1 mentions)
Microrna kit, by Qiagen (1 mentions)
Rnalater stabilization solution, by Thermo Fisher Scientific (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rna 6000 pico kit, by Agilent Technologies (1 mentions)
Rnasezap, by Merck Group (1 mentions)
Tecnai 12 biotwin tem, by Thermo Fisher Scientific (1 mentions)
4 protocols using rbma 200c
1
Quantification of RVLM TH+ Neurons
2
Transcriptomic Analysis of Brain Calcifications
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Mice were deeply anesthetized and transcardially perfused with ice-cold PBS. Brains were removed, placed in RNAlater Stabilization Solution (Thermo Fisher Scientific, catalog no. AM7020), and 1-mm coronal sections were cut using a brain matrix (RBMA-200C, World Precision Instruments). Calcifications were detected on the basis of their autofluorescence using a fluorescent stereomicroscope (Zeiss Axio Zoom.V16) and were surgically removed together with surrounding tissue. Cortical sections were also removed as examples of non–calcification-prone regions. RNA was isolated with a micro RNA kit (Qiagen) according to the manufacturer’s instructions. The concentration of RNA and sample purity were assessed using a 2100 Bioanalyzer (Agilent) and RNA 6000 Pico Kit (Agilent). RNA samples were poly(A)-enriched, and libraries were prepared using the Illumina TruSeq Stranded RNA kit. RNA was sequenced on an Illumina platform HiSeq 4000 at the Functional Genomic Center Zurich (UZH, ETH). The Illumina single-read approach (1 × 125 base pair) was used to generate raw sequencing reads with a depth of 20 million to 30 million reads per sample.
3
Hippocampus and mPFC Tissue Collection
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Adult male and female mice (week 10–12) were sacrificed via rapid decapitation between 9.30 and 10.30 AM. Dorsal hippocampus (upper third of the hippocampus on the dorsal side) and mPFC of both hemispheres were collected using a stainless-steel brain matrix (RBMA-200C, World Precision Instruments). Hippocampal and mPFC tissues were immediately processed into purified protein, in an RNase-free environment by use of RNaseZAP on all materials (Sigma-Aldrich). Pilot experiments in which we collected amygdala tissue with the use of micro-punches revealed that the amount of tissue collected in this manner was too low to allow reliable Western blot measurements.
As previously described (Sarabdjitsingh et al., 2010 (link); Loi et al., 2015 (link)), the samples were homogenized using a homogenizer (IKA® T10 basic) with ice-cold lysis buffer (RIPA) containing 1 M Tris, 1 M NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton, and 1 mM EDTA of pH 8. The samples were clarified by centrifugation for 20 min at 13,200 rpm at 4°C, then aliquoted and stored at −80°C for further use.
As previously described (Sarabdjitsingh et al., 2010 (link); Loi et al., 2015 (link)), the samples were homogenized using a homogenizer (IKA® T10 basic) with ice-cold lysis buffer (RIPA) containing 1 M Tris, 1 M NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton, and 1 mM EDTA of pH 8. The samples were clarified by centrifugation for 20 min at 13,200 rpm at 4°C, then aliquoted and stored at −80°C for further use.
4
Mouse Brain Ultrastructure Imaging via EM
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For electron microscopy, 10 ml of PBS and 40 ml of 2% PFA and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) were perfused through the left ventricle of an anaesthetized mouse. The brain was removed and further fixed by immersion for 3 h at room temperature (RT). An acrylic brain matrix for mouse (World Precision Instruments, RBMA-200C) and blades were used to obtain coronal slices, which were post-fixed in reduced 1% osmium tetroxide containing 1.5% potassium hexacyanoferrate. The tissue was dehydrated and embedded in epon and 60-nm ultrathin sections were cut on an ultramicrotome (UC6, Leica). The sections were counterstained with uranyl and lead and subsequently imaged on an electron microscope (Tecnai 12 Biotwin TEM, FEI). Representative pictures were documented in imaging plates (Ditabis, Pforzheim).
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