Cells were fixed in NBF, embedded in agarose gel (7 %) and then processed for paraffin embedding. Target retrieval solution (pH: 9.0; Dako, Glostrup, Denmark; Product No: S236884) heated for 40 min at 150 W in a microwave was used for antigen retrieval. Slides were then washed in washing buffer (Dako, Glostrup, Denmark; Product No: S3006) and incubated with a monoclonal antibody against GIRK1 (Abcam, Cambridge, UK; cat.No: 119246; 1:50; clone 3E11). For visualization, the EnVision + dual link reagent (rabbit/mouse horse radish peroxidase, Glostrup, Dako, Denmark; Product No: K406311) was used according to manufacturer’s protocols. Immunohistochemical staining was developed by incubation of sections with diaminobenzidine (DAB; Glostrup, Dako; Product No: K406511) as a chromogenic substrate. Slides were then washed in Dako wash buffer, counterstained with Meyer’s hematoxylin (from the pharmacy of the Medical University of Graz), rinsed in tap water, dehydrated and mounted with Entellan® (Merck, Darmstadt, Germany). Sections incubated without primary antibody served as negative controls.
Washing buffer
Manufactured by Agilent Technologies
Sourced in United States
Washing buffer is a solution used in various laboratory procedures to remove unwanted materials from samples or surfaces. It serves the core function of facilitating the cleaning and purification of samples, ensuring the removal of contaminants, excess reagents, or unbound molecules.
Automatically generated - may contain errors
Lab products found in correlation
Axioplan microscope, by Zeiss (2 mentions)
Low input quick amp labeling kit, by Agilent Technologies (2 mentions)
Dako wash buffer, by Agilent Technologies (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Entellan, by Merck Group (1 mentions)
Peroxidase block, by Agilent Technologies (1 mentions)
Dab substrate, by Agilent Technologies (1 mentions)
Axiocam icc3, by Zeiss (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Liquid permanent red, by Agilent Technologies (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Blocking solution, by Agilent Technologies (1 mentions)
Rnaeasy column purification, by Qiagen (1 mentions)
Scanner model g2505c, by Agilent Technologies (1 mentions)
Feature extraction software 10, by Agilent Technologies (1 mentions)
Genepix pro 6, by Molecular Devices (1 mentions)
6 protocols using washing buffer
1
Immunohistochemical Localization of GIRK1
2
Histological Analysis of Organs
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Immediately after the animals were sacrificed, organs were carefully excised and stored overnight in freshly prepared 4% formaldehyde solution. Afterwards, samples were embedded, thin-cut (thickness between 3 and 5 µM) and stained. Haematoxylin and eosin (H&E) stainings, periodic acid-Schiff reaction (PAS) stainings, and Giemsa stainings were prepared according to literature54 . Representative images were acquired on a Zeiss Axioplan microscope equipped with a AxioCam ICc 3 color camera with ZEN 2011 lite (BLUE EDITION) software. Intensity and contrast settings were adjusted for each organ but kept consistent between control and test sample. Foxp3-staining on embedded tissues was performed as follows: first, samples were thin-cut (3–5 µm); second, paraffin was melted (72 °C, 30 min) followed by Xylol (2 × 5 min), and alcohol treatment; third, samples were incubated at 120 °C in Tris–EDTA buffer for 5 min, followed by blocking with peroxidase-block (Dako); then, incubation with primary antibody (Foxp3 FJK-16 s, 1:50, 30 min at RT) and secondary antibody (anti-rat HRP, 30 min at RT) with intermittent washing steps for 5 min (Washing buffer from Dako); last, chromogenic detection solution was added for 10 min at RT (DAB+ substrate, Dako) followed by 1 min incubation with Hematoxylin (Merck) and washing steps.
3
Immunohistochemical Analysis of SARS-CoV-2 NP
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For immunohistochemistry (IHC), the FFPE lung tissues were sectioned at 6 μm, deparaffinised and rehydrated for staining preparation. The sections were then incubated in antigen unmasking solution vector (pH 6) at 95°C for 45 min for antigen retrieval. The endogenous tissue peroxidase was inactivated by 3% H2O2 at room temperature for 10 min, washed with washing buffer (Dako, USA), and digested with 20 μg/mL Proteinase K for 5 min at 37°C. To block non-specific reactions, the sections were incubated with 5% BSA (Sigma-Aldrich, USA) for 30 min. The sections were incubated with 1:400 dilutions of the SARS-CoV-2 Nucleoprotein/NP antibody, Rabbit Mab, Cat. No, 40143-R001 diluted in blocking buffer for 1 h at 37°C in a moist chamber. After washing with PBS, the sections were incubated with 1:25 polyclonal goat anti-rabbit immunoglobulin/AP secondary antibody, code No. D0487 diluted with blocking buffer for 1 h at 37°C in a moist chamber. The antibody was labelled with liquid permanent red (Dako, USA), and the signal was developed in LPR chromogen and substrate buffer for 20 min at room temperature in a dark moist chamber. All sections were counterstained with haematoxylin for 20s, mounted with mounting medium and covered by a coverslip.
4
Quantification of pERK1/2 Expression in Xenograft Samples
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Immunohistochemistry (IHC) was performed on available paraffin fixed tissue blocks from a xenograft experiment [10 (link)] to detect the expression of pERK1/2. Rehydration of tissue sections was performed in descending concentrations solutions of ethanol (96% to 70%). Antigen was retrieved by heating in a pressure cooker (1 mmol/L Tris-EDTA buffer, 120°C, 5 min). The slices were incubated in blocking solution (Dako, Glostrup, Denmark) to prevent nonspecific binding sites. Next pERK1/2 (1:2000) primary antibody was added to the slices and incubated for 30 min at room temperature. The sections were then washed with washing buffer (Dako) and incubated with EnVision + System horseradish peroxidase-linked secondary antibody (goat anti-rabbit, Dako) at room temperature for 30 min. Positive immunoreactivity was detected using diaminobenzidine + (Dako). Positive and negative IHC controls were included in this study. Percentage and intensity of positively stained epithelial cells (cytoplasmic versus nuclei) was quantified and scored by an expert pathologist (T.T.R.). Statistical analysis was performed using SPSS. Two tailed student t-test was performed by comparing 2 samples assuming that they have equal variances.
5
Whole Genome Oligo Microarray Analysis
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Microarray analyses were performed using the Whole Mouse Genome Oligo Microarray (Mouse GE 4x44K v2 Microarray Kit; G4846A) (Agilent Technologies, Santa Clara, CA) [27 ]. A full list of cDNAs is available online (www.agilent.com ). Protocols for sample preparation and hybridization of the mononuclear cells were adaptations of those in the Agilent Technical Manual. Briefly, Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using a Low Input Quick-AMP labeling kit (Agilent Technologies) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with a NanoDrop spectrophotometer. Thereafter, Cy3-labelled cRNA was fragmented at 60°C for 30 min. Fragmented cRNA samples were hybridized onto chips by means of 17 hr of incubation at 65°C with constant rotation, followed by a two-step microarray wash of 1 min in two washing buffers (Agilent Technologies). Hybridized microarrays were scanned in an Agilent Technologies Scanner (model G2505C), and the scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 026655_D_F_20100123) to obtain background subtracted and spatially de-trended Processed Signal intensities.
6
Custom Microarray-Based Gene Expression Analysis
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Sample preparation and hybridization was adapted from the Agilent technical manual (one colour). Each sample was hybridized individually. In short, first strand cDNA was transcribed from 200 ng of total RNA using T7-Oligo (dT) promoter primers. Samples were transcribed in vitro and Cy-3 labelled, all with the Low Input Quick Amp Labeling kit (Agilent Technologies, Inc., Santa Clara, CA, USA). The labelling reaction typically yielded between 4 and 5 μg of complementary RNA (cRNA) with a specific activity greater than 6. Fragmented cRNA samples were hybridized onto the customized ERA array (Ruiz-Alonso et al., 2013) , by incubation at 65°C for 17 h with constant rotation. The microarray was then washed in two steps of 1 min in two washing buffers (Agilent Technologies, Inc., Santa Clara, CA, USA). Hybridized microarrays were scanned in an Axon 4100A scanner (Molecular Devices, Sunnyvale, CA, USA), and data were extracted with the GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA, USA).
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