Liver tissue samples were dehydrated in ethanol, cleared in xylene, impeded in paraffin to form tissue blocks, which then sectioned (4-5 μm), finally the slides were stained by hematoxylin and eosin (H & E). Immunostaining was performed as previously described [18 (link), 24 (link)] using polyclonal rabbit anti-rat PCNA antibodies (1:500 dilution, Thermo-Scientific, USA) and goat anti-rabbit secondary antibody (1:1000 dilution, Dako, USA).
Goat anti rabbit secondary antibody
Manufactured by Agilent Technologies
Sourced in United States
The Goat anti-rabbit secondary antibody is a laboratory reagent used in various immunoassay techniques. It is designed to detect and bind to rabbit primary antibodies, allowing for the visualization and quantification of target analytes in samples.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Signalstain antibody diluent, by Cell Signaling Technology (1 mentions)
Digital pathology solution, by Philips (1 mentions)
Alx 210 333, by Enzo Life Sciences (1 mentions)
Acid phosphatase kit, by Merck Group (1 mentions)
Semi dry trans blot apparatus, by Bio-Rad (1 mentions)
Specific primary antibody, by Merck Group (1 mentions)
Sds page, by Bio-Rad (1 mentions)
X ray film, by Kodak (1 mentions)
Ecl plus western blotting detection reagent, by Cytiva (1 mentions)
Pt link, by Agilent Technologies (1 mentions)
Autostainer, by Agilent Technologies (1 mentions)
Interleukin 6 (il 6), by Abcam (1 mentions)
Tnf α, by Zeiss (1 mentions)
Il 17, by Abcam (1 mentions)
Phospho smad3, by Rockland Immunochemicals (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Phospho p65, by Cell Signaling Technology (1 mentions)
11 protocols using goat anti rabbit secondary antibody
1
Liver Tissue Histopathology Evaluation
2
GAP43 Immunohistochemistry in Paraffin Sections
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Four-micrometre-thick paraffin sections were cut, mounted on slides, deparaffinized in xylene, rehydrated in decreasing ethanol dilutions and incubated in 3% hydrogen peroxide buffer for 30 min. After blocking of endogenous peroxidase activity, the slides were boiled with citrate buffer (pH 6.0) in a pressure cooker for 90 s, followed by blocking of non-specific binding sites with 5% blocking serum for 30 min at room temperature. The sections were then incubated with rabbit anti-GAP43 antibody (Huabio, Hangzhou, China) at a dilution of 1:1200 overnight at 4 °C. Afterwards, 1:2000 diluted goat anti-rabbit secondary antibody (Dako, Glostrup, Denmark) was applied for 30 min. The reaction products were visualized using 3,3′-diaminobenzidine (DAB; Dako), and the sections were lightly counterstained with hematoxylin and mounted. The results were evaluated by two experienced pathologists and photographed.
3
Immunohistochemistry of Angiogenic Markers
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The expression of CD31, α‐SMA, and VEGF were investigated by immunohistochemistry, which was conducted as described previously.25 Briefly, after antigen retrieval, the nonspecific antigens were blocked with 5% goat serum at 37°C for 30 min. The sections (4 μm) were exposed to rabbit anti‐CD31, α‐SMA, and VEGF primary antibody (all at 1:100, Abcam, Cambridge, UK) at 37°C for 60 min, respectively. After 3 washes with phosphate‐buffered saline (PBS), the sections were incubated with goat anti‐rabbit secondary antibody (1:200; Dako, CA, USA) at 37°C for 60 min, developed and counterstained with 3, 3′‐diaminobenzidine tetrahydrochloride (DAB) solution. The areas stained with brown color indicated positive staining under an optical microscope.
4
Immunohistochemical Scoring of Met Expression
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All slides were processed under identical conditions using standard protocols. The antibody was diluted at 1:150 (Met (D1C2) XP® Rabbit mAb ,Cell Signaling Technology, USA) using SignalStain antibody diluent (Cell Signaling Technology, USA), was applied to slides for 16 h at 4°C. Goat anti-rabbit secondary antibody (DAKO) was incubated for 15 min and then following by the routine staining procedure. The IHC score was classified as 0 to 3+according to staining strength in membrane: no staining or <10% tumor cells (score 0), faint staining in >10% (score 1+), moderate staining in >10% (score 2+), and strong staining in >10% (score 3+). Score 0/1+ and 2+/3+ were regarded as negative and positive respectively.
5
Histological Analysis of Bone Remodeling
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Femurs were fixed with 4% phosphate buffered formalin, decalcified for three weeks in 10% EDTA, paraffin embedded and cut into 5 µm sections. Sections were stained with haematoxylin and eosin (HE) following a standard protocol. To visualise osteoclasts, sections were stained for TRAP using an acid phosphatase kit (Sigma‐Aldrich), and counterstained with a Light Green solution. Osteoblasts were stained using a primary anti‐osteocalcin antibody (ALX‐210–333, 1:1000; Enzo Life Sciences) in combination with a goat anti‐rabbit secondary antibody (Dako). Sections were counterstained with haematoxylin. Slides were digitalized with Philips Digital Pathology Solutions (PHILIPS Electronics) for morphological measurement, and both osteoclast and osteoblast surface areas were quantified using TrapHisto open‐source software (van 't Hof, R. J., Rose, L., Bassonga, E., & Daroszewska, A., 2017 (link)).
6
Immunohistochemical Analysis of CD68 and TGF-β
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For the immunohistochemical analysis, the sections were deparaffinized, washed with PBS three times, and blocked with 5% serum for 30 minutes. Then, the slides were treated with rabbit anti-CD68 primary antibody (1:100; Santa Cruz, USA) and rabbit anti-TGF-β primary antibody (1:100; Abcam, UK) at 4 °C overnight. The slides were further incubated with goat-anti-rabbit secondary antibody (1:200; Dako, CA, USA) at 37 °C for 30 minutes, developed with 3,30-diaminobenzidine tetrahydrochloride (DAB) solution, and counterstained with hematoxylin. Brown color indicates positive staining under an optical microscope.
7
Protein Expression Quantification by Western Blot
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Proteins of tissue homogenates were separated on SDS-PAGE (Bio-Rad, Copenhagen, Denmark). After transfer with a semi-dry transblot apparatus (Bio-Rad, Copenhagen, Denmark) the membranes were blocked with 1% nonfat dry milk for one hour at room temperature. The membranes were each incubated with the specific primary antibody (Millipore, Schwalbach, Germany) at 4°C overnight followed by washing three times in wash buffer I for 15 min at room temperature. They were then incubated in goat anti-rabbit secondary antibody (DAKO, Hamburg, Germany). After washing with horseradish peroxidase complex (Amersham) for one hour, each protein was visualized using DAB solution [1%DAB and 0.75% H2O2 in 0.1M PB (pH 7.4)]. Protein content was quantified by densitometric measurement. Actin was used as loading control.
8
Profiling P. berghei Protein Expression
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For Western analysis mixed P. berghei blood stages (2×107 parasites) were resuspended in 2× SDS gel-loading buffer (100 mM Tris-HCl pH 6.8; 200 mM dithiothreitol; 4% SDS; 0.2% bromophenol blue; 20% glycerol) with prior addition of 1 µl of PMSF (Ci = 100 mM, Cf = 1 mM). The samples were run on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane, which was subsequently blocked with 5% milk/0.1% Tween in PBS, probed with the PfTERT primary antibody [29] (link) in 5% milk/0.1% Tween in PBS (rabbit, 1∶500, courtesy of A. Scherf), washed 3× with 0.1% Tween/PBS and probed with an HRP-labelled secondary goat anti-rabbit antibody (Dako) in 5% milk/0.1% Tween in PBS (1∶5000). Same procedure was used for the control antibody against enolase (PBANKA_121430; rabbit, 1∶1000, Biogenes). The complex was visualised using the ECL Plus™ Western blotting detection reagents (Amersham Biosciences), and X-ray film (Kodak).
9
Quantifying PEG in Tissue Samples
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Paraformaldehyde fixed muscle and tumor tissues collected before injection and at 30 minutes and 2–4 h post-dose were paraffin embedded and thereafter cut (4 µm) and positioned on glass tissue slides. Prior to staining, the slides were placed in an antigen-retrieval solution using an automated pre-treatment module (PT-Link; Dako, Glostrup, Denmark). Slides were stained for PEG in an automated immunohistochemistry robot (Autostainer; Dako) using a primary rabbit anti-PEG-B-47 antibody (Abcam, Cambride, MA) at a concentration of 0.6 µg/ml and a secondary goat-anti rabbit antibody (Dako) at 1∶2000 dilution. Slides were counterstained with hematoxylin, dehydrated and mounted. The stained slides were digitized and total PEG-staining was quantified with the Visomorph software (Visiopharm, Hoersholm, Denmark). Findings in staining patterns were confirmed by a trained pathologist.
10
Immuno-electron Microscopy of Nephrin
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The paraformaldehyde-glutaraldehyde-fixed 1-mm blocks of renal cortex were washed with PBS, dehydrated through graded ethanol, and embedded in Epon. Ultrathin sections were transferred to nickel grids and blocked with 1% bovine serum albumin and 1% normal goat serum in PBS. Sections were incubated with a primary polyclonal goat anti-human nephrin antibody against the intracellular domain of nephrin (1:50; R&D Systems), then with a secondary gold-conjugated (10 nm) rabbit anti-goat secondary antibody (1:100; DAKO, DK). Sections were postfixed with 1% glutaraldehyde, contrasted with uranyl acetate and lead citrate, and observed under an electron microscope and photographed for detailed analysis.
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