Immunohistochemistry is performed on the paraffin-embedded (5 µm) sections. Paraffin embedded sections are deparaffinised and rehydrated as previously described in McGovern et al. (2013) (link). HSE and MSE skin sections are subjected to heat-mediated antigen retrieval treatment using either sodium citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) in a decloaking chamber (Biocare Medical, USA) as described in Table 2 . All skin sections are washed in phosphate buffered saline followed by immunostaining using MACH 4™ Universal HRP polymer kit (Biocare Medical). The temperature and time varies for each marker, as outlined in Table 2 . The primary antibody for each protein is diluted in DaVinci Green diluent (Biocare Medical) to concentrations specified in Table 2 , and these sections are incubated with the primary antibody for the time specified in Table 2 . All the sections are finally counterstained using Gill’s haematoxylin (HD Scientific), dehydrated, mounted and imaged as described in ‘Histological Analysis’.
Da vinci green diluent
Manufactured by Biocare Medical
Sourced in United States
The Da Vinci Green Diluent is a laboratory reagent used for the dilution of samples prior to analysis. It is a non-toxic, aqueous solution that is designed to maintain the integrity of the sample during the dilution process. The core function of the Da Vinci Green Diluent is to provide a consistent and reliable means of preparing samples for further testing or analysis.
Automatically generated - may contain errors
Lab products found in correlation
Decloaking chamber, by Biocare Medical (3 mentions)
Methyl green, by Vector Laboratories (2 mentions)
Background sniper, by Biocare Medical (2 mentions)
Rodent block m, by Biocare Medical (2 mentions)
Dab solution, by Agilent Technologies (2 mentions)
Target retrieval solution, by Agilent Technologies (2 mentions)
Meyer s hematoxylin, by Merck Group (2 mentions)
Mach4 universal hrp polymer kit, by Biocare Medical (1 mentions)
Alexa fluor 555, by Merck Group (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 680 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Rabbit anti tdp43, by Cell Signaling Technology (1 mentions)
Fluoro gel 2 with dapi, by Electron Microscopy Sciences (1 mentions)
Mouse anti neun clone a60, by Merck Group (1 mentions)
Alexa fluor 488, by Cell Signaling Technology (1 mentions)
Mouse anti gfap, by Cell Signaling Technology (1 mentions)
Rabbit anti iba1, by Fujifilm (1 mentions)
Pepsin solution, by Thermo Fisher Scientific (1 mentions)
Rat igg1, by Thermo Fisher Scientific (1 mentions)
Dual endogenous block, by Agilent Technologies (1 mentions)
13 protocols using da vinci green diluent
1
Immunohistochemistry of Skin Sections
2
Multiplex Immunofluorescence Staining
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Slides were acclimated to RT and rinsed in distilled water. Heat-induced epitope retrieval was performed at 120°C using pH 6.0 buffer (Epredia, TA-135-HBL). The tissues were then blocked in 10% NGS for 1 hr. Primary antibodies were applied overnight at 4°C: Mouse Anti-NeuN (clone A60) conjugated to Alexa Fluor 555 (Millipore, MAB377A5), and Rabbit Anti-TDP-43 (Cell Signaling, 89789) or Rabbit anti-Iba1 (Wako, 019-19741) and mouse anti-GFAP conjugated to Alexa Fluor 488 (Cell Signaling, 3655) (Supplementary file 4 ). After rinsing, secondary antibodies were applied for 2 hr at RT: Alexa Fluor 488 Goat Anti-Rabbit IgG (Invitrogen, A32731) or Alexa Fluor 680 Goat anti-Rabbit IgG (Invitrogen 32734). All antibodies were diluted using Da Vinci Green Diluent (Biocare Medical, PD900L). Finally, slides were coverslipped using Fluorogel II with DAPI (Electron Microscopy Sciences, 17985-50).
3
Immunohistochemical Detection of Eosinophil MBP
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To detect the eosinophil MBP in lung tissues, sections were deparaffinized and rehydrated. Endogenous peroxidase and alkaline phosphatase were blocked by treating the sections with a dual endogenous block (Dako, Carpinteria, CA) for 10 minutes. After washing with PBS, the sections were incubated with a pepsin solution (Life Technologies, Digest All 3, Grand Island, NY) for 10 minutes to unmask the antigenic sites. After another wash, sections were blocked with Background Sniper (BioCare Medical, Concord, CA) for 5 minutes. Sections were then washed and incubated overnight at 4°C with 4 μg/mL of either rat IgG1 (eBiosciences, San Diego, CA) or rat anti-mouse MBP (kindly provided by Dr. James J. Lee, Mayo Clinic Arizona, Clone MT-14.7), which was diluted in Da Vinci Green Diluent (BioCare Medical). Staining was visualized using a rat-on-mouse alkaline phosphatase-polymer detection kit (BioCare Medical) and a fast red chromogen kit (BioCare Medical), as per the manufacturer’s instructions. Sections were counterstained with methyl green (Vector Laboratories, Burlingame, CA) and mounted with Permount (Fisher, Waltham, MA).
4
Immunohistochemical Analysis of Gal-3 in Tumor Samples
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Tumor sections were deparaffinized, rehydrated and heated in a steamer for 30 minutes for antigen retrieval in citrate buffer pH 6.0. After cooling, sections were incubated with peroxidase block for five minutes, and then serum-free protein block (DAKO, X0909) for 20 minutes. Slides were then incubated at room temperature for one hour with mouse monoclonal Gal-3 antibody (Leica microsystems, NCL-GAL3, 1:250) diluted in Da Vinci green diluent (Biocare Medical, PD900). The slides were incubated with Envision anti-mouse HRP-labeled polymer (DAKO, K4000) for 30 minutes. The slides were visualized with diaminobenzidine (DAKO), washed in distilled water, hematoxylin counterstained, dehydrated and mounted. Positive and negative controls were included in each panel of staining. All the slides were evaluated and scored by a pathologist (X.L.) blinded to clinical data. All the scoring was performed twice with at least three-day interval. A subset of samples for each marker was also reviewed by a second pathologist (S.R.) unaware of other data. H-Scores were calculated by multiplying staining intensity (1, 2, 3) with staining percentage (0% –100%).
5
Immunohistochemical Staining of GFP Proteins
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Sections (three per each 6-mm block, with a separation of 2 mm) were washed three times in PBST for 5 minutes each time and then treated with 1% H2O2 for 20 minutes. Sections were incubated in Sniper blocking solution (http://biocare.net/product/background-sniper/ ) for 30 minutes at room temperature and then incubated overnight with the primary anti-GFP antibody (https://www.lifetechnologies.com/ ) diluted 1:1,000 in Da Vinci Green Diluent (http://biocare.net/ ). After three rinses in PBS containing 0.1% Tween-20 (PBST) for 5 minutes each time, sections were incubated in Mach 2 horseradish peroxidase (HRP) polymer (http://biocare.net/ ) for 1 hour and then washed three times before colorimetric development with 3,3’-diaminobenzidine. Immunostained sections were counterstained with cresyl violet, and mounted on slides and sealed with Cytoseal (http://www.thermoscientific.com/ ).
6
Immunohistochemical Analysis of Flavivirus NS1
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Liver lobes were cut and fixed in 10% formalin overnight, embedded in paraffin and sections stained with hematoxylin and eosin (H & E). For immunohistochemistry, paraffin sections were rehydrated via an alcohol series, placed in Target Retrieval Solution, pH 9.0 (Dako/Agilent, Santa Clara, CA, USA) for 20 min at 100 °C using the Decloaking chamber (Biocare Medical, Pacheco, CA USA), blocked using Rodent Block M (Biocare Medical) overnight and Biocare Medical Background Sniper (with 1% BSA, 20% donkey serum, 20% goat serum) for 15 min, and then stained overnight at room temperature with anti-flavivirus NS1 monoclonal antibody 4G4 (Mozzy mAbs; https://eshop.uniquest.com.au/mozzy-mabs/ ) as tissue culture supernatant diluted in Da Vinci Green Diluent (Biocare Medical). After washing, endogenous peroxidase was blocked by incubation in 2% hydrogen peroxide for 10 min and ImmPRESS HRP (horse) Anti-Mouse IgG (Peroxidase) Polymer Detection Kit (Vector Laboratories, Burlingame, CA, USA) added for 1 h. After washing, signal was developed using Vector NovaRED Peroxidase (HRP) Substrate Kit (LS Bio, Seattle, WA, USA), with light haematoxylin counter-staining.
7
Immunohistochemical Analysis of B7-H3 and Ki-76
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Antigen retrieval was performed on 4 µm sections with heat-induced epitope retrieval in a decloaking chamber (Nexgen, Biocare Medical, Concord, CA, USA) with sodium citrate buffer (0.01 M, pH 6.0) at 110 °C for 20 min. The reagents from the MACH1 Universal HRP detection Kit (Biocare Medical, LLC, Concord, CA, USA, #M1U539 L10) was used for immunohistochemical detection. MACH1 sniper blocking reagent was used to block non-specific staining for 30 min. Primary antibodies against B7-H3 (1:200; Cell Signaling, #D9M2L/#14058, Beverly, MA, USA) and Ki-76 (1:100; Dako, #M7240, Santa Clara, CA, USA) were diluted in Biocare Da Vinci Green Diluent (Biocare Medical, #PD900, Concord, CA, USA) and incubated overnight at room temperature. The slides were treated with MACH1 secondary antibody conjugated with MACH1 Horse-Radish Peroxidase polymer for 30 min at room temperature. MACH1 diaminobenzidine substrate was applied for 5 min and the slides were counterstained with hematoxylin.
8
Immunohistochemical Analysis of BRCA1 and 53BP1
9
Immunohistochemical Analysis of CD34+ Cells
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Tissues were dissected and fixed in 10% neutral buffered formalin (Fisher Scientific, SF99-4) for 24 hours at room temperature and processed in the Hypercenter XP (Thermo Fisher) using a standard dehydration protocol before paraffin embedding and microtome sectioning into 4 μm sections. Tissue sections were deparaffinized with xylene substitute (Thermo Fisher) before rehydration with gradients of alcohol. Heat-induced antigen retrieval was performed in Rodent Decloaker solution (Biocare Medical, RD913) in the Decloaking chamber (Biocare Medical). Non-specific interaction of primary antibodies with the tissue was blocked by Rodent block M (Biocare Medical, RBM961). Tissues were then stained with Rabbit anti-CD34 (Abcam, ab81289) at 1:3000 dilution in Da Vinci Green diluent (Biocare Medical, PD900) overnight at 4° C. The tissue section was stained with Rabbit-on-rodent-HRP polymer (Biocare Medical, RMR622). The slides were treated with Vector ImmPact® VIP peroxidase substrate (Vector labs, SK-4605) for 10 minutes at room temperature. Methyl green (Vector labs, H-3402) was used to counterstain.
10
Immunohistochemical Profiling of Muscle Markers
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The sections were labelled with the following monoclonal antibodies: Bcl-2, CD68, CD163, FAS, Granzyme B, HSP70, Merosin 80, MyHC-fast, MyHC-slow and Spectrin (for details see Table 2 ). Primary antibodies were diluted in Da Vinci Green diluent (BiocareMedical, CA, USA) and incubated at room temperature in a humid chamber. Tris-buffered saline (TBS; 50 mM), pH 7.4, was used as washing buffer. Bound antibodies were detected using the Novolink Polymer Detection System (Leica Biosystems Nussloch GmbH, Germany), according to the manufacturer’s protocol. Novocastra DAB enhancer was used to improve the Bcl-2 visualization. Negative controls were performed by replacing the primary monoclonal antibody with IgG1 isotype control (X0931, Dako Denmark A/S) or by omitting the primary antibody.
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