Single-molecule RNA in situ hybridization SARS-CoV-2 RNA was detected by using RNAscope technology (Advanced Cell Diagnostics, Inc., Newark, CA, USA), an RNA in situ hybridization (ISH) technique. Paired double Z oligonucleotide probes were designed for hybridization to the target RNA by using custom software. The RNAscope 2.5 LS Probe V-nCoV2019-S (catalog number 848568; Advanced Cell Diagnostics) was used. The RNAscope 2.5 LSx Reagent Kit-Brown (Advanced Cell Diagnostics) in combination with a BOND-III Automated stainer (Leica Biosystems, Wetzlar, Germany) was used to process the samples according to the manufacturer’s instructions. The FFPE tissue section samples were prepared according to the manufacturer’s recommendations. The RNA integrity of each sample was evaluated with a probe designed for hybridization specifically to the ubiquitin C and cyclophilin B housekeeping genes. The negative control background staining was evaluated using a probe specific to the bacterial dapB gene. Each punctate dot signal representing a single target RNA molecule could be detected with standard light microscopic analysis.
Bond 3 automated stainer
Manufactured by Leica
Sourced in Germany, United States
The BOND-III is an automated stainer from Leica designed for IHC (immunohistochemistry) and ISH (in-situ hybridization) sample preparation. It offers automated slide processing including deparaffinization, epitope retrieval, staining, and coverslipping. The BOND-III provides standardized, high-quality results through its programmable, walk-away functionality.
Automatically generated - may contain errors
Lab products found in correlation
Glial fibrillary acidic protein (gfap), by Agilent Technologies (2 mentions)
Rnascope 2.5 lsx reagent kit brown, by Advanced Cell Diagnostics (1 mentions)
Rnascope 2.5 ls probe 5 ncov2019 s, by Advanced Cell Diagnostics (1 mentions)
Rnascope technology, by Advanced Cell Diagnostics (1 mentions)
Ab61034, by Abcam (1 mentions)
Bond polymer refine detection kit, by Leica (1 mentions)
Envision flex hrp system, by Agilent Technologies (1 mentions)
Ix73 microscope, by Olympus (1 mentions)
β catenin clone 14, by BD (1 mentions)
D1306, by Thermo Fisher Scientific (1 mentions)
Anti panck, by Gene Tech (1 mentions)
Gm351507, by Gene Tech (1 mentions)
Ab281583, by Abcam (1 mentions)
10 protocols using bond 3 automated stainer
1
Single-molecule SARS-CoV-2 RNA Detection by RNAscope
2
Placental Hofbauer Cell Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical staining with NovocastraTM Liquid Mouse Monoclonal Antibody CD163 (10D6 clone) (Leica Biosystems, Buffalo Grove, IL, USA) was used to identify Hofbauer cells in the placenta, combined with RNAscope 2.5 LS Probe V-nCoV2019-S. The double staining was performed on BOND-III Automated stainer (Leica Biosystems) according to the manufacturer’s instructions.
3
Quantifying DKK1 Expression in Liver Tumors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DKK1 protein expression was determined using a DKK1 antibody (ab61034; Abcam, Cambridge, MA, USA) in liver FFPE tumor sections from six HCC patients and the RGC risk subgroups (HRT and LRT) in accordance with a standard immunohistochemistry (IHC) protocol. Automated hematoxylin and diaminobenzidine staining (IHC DAB1 Leica Bond III) using a Leica Bond III Automated Stainer (Leica Biosystems, Wetzlar, Germany) was performed using the protocol: (a) dewaxing; (b) pretreatment; and (c) IHC staining. The dewaxing step was performed at 72 °C, followed by pretreatment (unmasking) with the Bond epitope retrieval 2 (ER2) solution, with washing steps being performed using absolute alcohol and Bond Wash Solution. IHC staining was performed using the primary antibody and the Bond™ Polymer Refine Detection kit (Leica Biosystems) was used for subsequent detection. After staining, the slides were dehydrated in absolute alcohol, cleaned with xylene and mounted with DEPEX mounting media (WVR International, Radnor, PA, USA).
4
Detecting SARS-CoV-2 in Lung Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In each patient, the FFPE RT-PCR positive lung sample with the lowest Ct value was also analyzed with IHC and ISH.
For SARS-CoV-2 immunohistochemistry, we used the monoclonal antibody (1A9, Genetex Inc., Irvine CA, United States) against the spike protein of SARS-Cov/SARS-CoV-2 in 1:100 dilution on 3 μm slides obtained from paraffin blocks. Antigen retrieval was performed with 10 mM citrate buffer (pH 6.0). The staining was visualized with the EnVision system FLEX/HRP (Agilent, Santa Clara, CA, United States).
RNA ISH for SARS-CoV-2 was performed using the RNAscope® SARS-CoV-2 probes for the SARS-CoV-2 S gene encoding the spike protein (catalog #848561, Advanced Cell Diagnostics, Inc., Hayward, CA, United States) on a Leica Bond III automated stainer (Leica Biosystems, Wetzlar, Germanyı), according to the manufacturer’s instructions. Briefly, 4-μm formalin-fixed and paraffin-embedded tissue sections were pre-treated with heat and protease prior to hybridization. Tissue sections were hybridized separately with the target probe to detect infected cells and with the positive and negative control probes. Specific staining signals were identified as brown, punctate dots present in the cytoplasm.
For SARS-CoV-2 immunohistochemistry, we used the monoclonal antibody (1A9, Genetex Inc., Irvine CA, United States) against the spike protein of SARS-Cov/SARS-CoV-2 in 1:100 dilution on 3 μm slides obtained from paraffin blocks. Antigen retrieval was performed with 10 mM citrate buffer (pH 6.0). The staining was visualized with the EnVision system FLEX/HRP (Agilent, Santa Clara, CA, United States).
RNA ISH for SARS-CoV-2 was performed using the RNAscope® SARS-CoV-2 probes for the SARS-CoV-2 S gene encoding the spike protein (catalog #848561, Advanced Cell Diagnostics, Inc., Hayward, CA, United States) on a Leica Bond III automated stainer (Leica Biosystems, Wetzlar, Germanyı), according to the manufacturer’s instructions. Briefly, 4-μm formalin-fixed and paraffin-embedded tissue sections were pre-treated with heat and protease prior to hybridization. Tissue sections were hybridized separately with the target probe to detect infected cells and with the positive and negative control probes. Specific staining signals were identified as brown, punctate dots present in the cytoplasm.
5
Immunohistochemical Characterization of Lung Cancers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Organoids and their corresponding parental tumors were fixed in 4% paraformaldehyde, followed by paraffin embedding, sectioning, deparaffinization, dehydration, and hematoxylin and eosin staining. Immunohistochemistry (IHC) was performed using antibodies targeting the thyroid transcription factor (TTF-1), Cytokeratin 7 (CK7), and Napsin A for ADC; Cytokeratin 5/6 (CK5/6), P40, and P63 for SCC; TTF-1, synaptophysin (Syn), chromogranin A (CgA), and CD56 for SCLC. Green Chromogen and the BOND IHC polymer detection kit were used for color development. The IHC staining was performed using the BOND-III automated stainer (Leica, Wetzlar, Germany). Images were captured using the IX73 microscope (Olympus, Tokyo, Japan).
6
Comprehensive Immunohistochemical Tissue Analysis
7
Immunohistochemical Profiling of Brain Tissue
8
Immunohistochemical Analysis of β-Catenin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical staining for β-catenin was performed on the Leica BOND III Automated stainer. Briefly, 4 μm TMA sections were deparaffinized with xylene and ethanol. Antigen retrieval was performed using citrate buffer (pH 6.0) for 20 min. Sections were incubated with clone 14/β-catenin (BD Transduction Lab, Franklin Lakes, NJ) diluted 1:20 for 15 min. External positive and negative controls were performed.
β-catenin staining was scored based on the intensity of cytoplasmic staining using a 3-point scale from 0 to 3: (0: absent; 1: weak; 2: moderate; 3: intense). Scoring was performed by a board-certified pathologist (AvZ) blinded to patient outcomes. The mean score of the multiple tumor samples was utilized for statistical analysis.
β-catenin staining was scored based on the intensity of cytoplasmic staining using a 3-point scale from 0 to 3: (0: absent; 1: weak; 2: moderate; 3: intense). Scoring was performed by a board-certified pathologist (AvZ) blinded to patient outcomes. The mean score of the multiple tumor samples was utilized for statistical analysis.
9
Comprehensive Tumor Microenvironment Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To comprehensively describe the tumor immune microenvironment, we used the serial FFPE slides of the biopsy specimens collected at baseline or during the combination in multiplex immunofluorescence staining. The FFPE slides were 4 μm thick so monolayer cells could be identified in the following imaging analysis. The multiplex immunofluorescence staining was automatically performed in a Bond III automated stainer (Leica, USA). The TSA 5-color kit (#D110051-50T) and TSA 670 (#D110016-100T) were bought from Yuanxibio, Shanghai, China. Two staining panels were applied. The staining order was as follows: panel 1—anti-CD4 (#YX32005, Yuanxibio, 1:300)/TSA 620, anti-CD8 (#YX63005, Yuanxibio, 1:300)/TSA 670, anti-panCK (#GM351507, GeneTech, Shanghai, China, 1:6)/TSA 520, and anti-PD-1 (#10377-MM23, Sino Biological, Beijing, China, 1:200)/TSA 570; panel 2—anti-PD-L1 (#13684, CST, Danvers, USA, 1:800)/TSA 570, anti-panCK (#GM351507, GeneTech, 1:6)/TSA 520, anti-CD11c (#45581, CST, 1:300)/TSA 620, and anti-CD68 (#GM087602, GeneTech, ready-to-use)/TSA 670. To visualize the cell nuclei, the tissue was stained with 4′,6-diamidino-2-phenylindole (D1306; Thermo Fisher, Waltham, USA).
10
CD31 Immunohistochemical Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the presence and expression of CD31, slides were dried and incubated in cold acetone for 20 min and then washed in PBS. Staining was performed with a Leica Bond-III Automated Stainer according to manufacturer’s instructions using the primary antibody for CD31 (ab281583, Abcam, dilution 1:200). The immunostaining area of CD31 was measured using ImageJ. The red channel was separated from blue and converted to grayscale, and the background was subtracted. The values of the monochromatic images were then measured and expressed as percentage of area. To quantify the CD31 staining regions, three areas from each section (ROIs) were selected from non-necrotic areas, and their average was calculated per each slide. One tumor was excluded from the analysis according to Grubb’s test.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!