All chemicals were purchased from Sigma-Aldrich, unless otherwise stated. Seventy-micrometer brain sections from two mice were rinsed in TBS 4 × 8 min before and after incubation in 10 % MeOH and 3 % H2O2 (Merck Millipore) in TBS for 10 min. Sections were incubated in 1 % blocking reagent (Roche Diagnostics) for 1 h followed by primary antibody incubation with anti-MFSD1 (1:1000) and anti-MFSD3 (1:1000) diluted in supermix (0.25 % gelatine, 0.5 % Triton X-100 in TBS) overnight at 4 °C. Sections were rinsed in TBS 2 × 1 + 4 × 8 min and incubated in secondary antibody (biotinylated goat-anti-rabbit IgG (H + L), Vector laboratories) diluted 1:400 in supermix for 1 h. Sections were rinsed in TBS 5 × 8 min before and after incubation in ABC kit (Reagent A, Reagent B (Vectastain, Vector Laboratories) and diluted 1:800 in supermix for 1 h. Sections were incubated in 0.05 % 3.3 diaminobenzidine tetrahydrochloride, 0.35 % NiCl and 0.015 % H2O2 and rinsed 4 × 5 min in TBS before mounted on gelatinized microscope slides (Menzel Gläser). Samples were dehydrated in 70 and 95 % EtOH for 5 min, 100 % EtOH (Solveco) for 10 min prior xylene incubation for 20 min. Slides were mounted in DPX Mounting for histology with micro cover slides Superfrost Plus (Menzel Gläser). Sections were analysed using a Mirax Pannoramic midi scanner with the Pannoramic Viewer software version 1.15.4 RTM (3dHistech).
Biotinylated goat anti rabbit igg h l
Manufactured by Vector Laboratories
Sourced in United States
Biotinylated goat anti-rabbit IgG (H+L) is a secondary antibody that binds to primary antibodies raised in rabbits. It is conjugated with biotin, which can be used to detect and amplify the signal from the primary antibody in various immunoassays and detection techniques.
Automatically generated - may contain errors
Lab products found in correlation
Blocking reagent, by Roche (1 mentions)
Pannoramic viewer software, by 3DHISTECH (1 mentions)
Hematoxylin and eosin staining, by Merck Group (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Harris hematoxylin, by Bio-Optica (1 mentions)
Vectastain elite abc reagent, by Vector Laboratories (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Dako liquid dab substrate chromogen system, by Agilent Technologies (1 mentions)
4 protocols using biotinylated goat anti rabbit igg h l
1
Immunohistochemical Detection of MFSD1 and MFSD3
2
NUAK1 Immunostaining Protocol
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Primary antibody used for immunostaining was the NUAK1 polyclonal antibody (sc-130117, 1 : 100 dilution; Santa Cruz, Dallas, TX, USA). Briefly, slides were deparaffinised in xylene and transferred through two changes of 100% ethanol. For antigen retrieval, the slides were boiled in a pressure cooker at maximum heat for 2 min containing 0.01 mol l−1 sodium citrate (pH 6.0) and cooled for 30 min at room temperature. Endogenous peroxidase activity was blocked in 0.3% H2O2 for 10 min at 37 °C. After incubation, the slides were washed 3 times in PBS for 3 min each. Then slides were incubated with the primary antibody overnight at 4 °C. Routine controls using PBS instead of the primary antibody were included to verify specificity. After washing with PBS, the bound primary antibody was detected by Biotinylated Goat Anti-Rabbit IgG (H+L) (1 : 200 dilution; Vector Laboratories, Burlingame, CA, USA) and the chromogenic substrate 3,3-diaminobenzidine tetrahydrochloride (DAB). The specimens were counterstained with haematoxylin, mounted, and examined by light microscopy (Olympus IX71, Tokyo, Japan).
3
Immunohistochemical Analysis of Tumor Sections
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Formalin-fixed, paraffin-embedded tumour sections were evaluated by hematoxylin-and-eosin staining (Sigma Aldrich). For immunohistochemistry, sections were retrieved in citrate buffer (pH 6) for 15 min and incubated overnight at 4 °C with anti-cytokeratin 7 (CK-7) SP52 antibody (Ventana, Tucson, AZ, USA) or Cleaved Caspase-3 (Asp175) (5A1) antibody (Cell Signaling Technology, Danvers, MA, USA). Slides were then incubated with a biotinylated goat anti-rabbit IgG (H+L) (1:200; Vector Laboratories, Burlingame, CA) for 1h at room temperature. Negative controls were run simultaneously omitting primary antibody while incubating with buffer. Staining was performed and visualized by 3,3'-diaminobenzidine (DAB, Vector Laboratories). All slides were counterstained with Harris hematoxylin (Bio Optica, Milan, Italy).
4
Histological Analysis of Implanted Devices
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At the end of the experiment (56 days after MSC implantation, five animals; 16 months, one animal), all transplant devices were excised from the animals, fixed overnight in 10 % buffered formalin (pH 7.4) at 4 °C and embedded in paraffin blocks. Serial sections (4 μm) were cut and stained with haematoxylin and eosin (H&E), Masson’s trichrome and elastic van Gieson stain.
Immunohistochemical detection of CD31 (rabbit polyclonal, Acris Antibodies GmbH, Germany) was performed on 4-μm-thick paraffin sections using a three-step indirect method. After deparaffinisation in xylene and rehydration in graded ethanol, antigen retrieval (EDTA buffer, pH 8), endogenous peroxidase and endogenous biotin blocking, sections were covered with normal goat serum (Vector Laboratories, USA) for 20 min. Primary antibody anti-CD31 was applied overnight at 4 °C; the antibody was detected by biotinylated goat anti-rabbit IgG (H+L) (Vector Laboratories, Burlingame, CA, USA), and then the sections were incubated with R.T.U. Vectastain Elite ABC Reagent (Vector Laboratories, USA) for 30 min. Finally, visualisation was performed with the Dako Liquid DAB+ Substrate-Chromogen System (Dako, Denmark) and counterstaining with Harris’ haematoxylin.
Each slide was viewed using standard light microscopy (Olympus BX41).
Immunohistochemical detection of CD31 (rabbit polyclonal, Acris Antibodies GmbH, Germany) was performed on 4-μm-thick paraffin sections using a three-step indirect method. After deparaffinisation in xylene and rehydration in graded ethanol, antigen retrieval (EDTA buffer, pH 8), endogenous peroxidase and endogenous biotin blocking, sections were covered with normal goat serum (Vector Laboratories, USA) for 20 min. Primary antibody anti-CD31 was applied overnight at 4 °C; the antibody was detected by biotinylated goat anti-rabbit IgG (H+L) (Vector Laboratories, Burlingame, CA, USA), and then the sections were incubated with R.T.U. Vectastain Elite ABC Reagent (Vector Laboratories, USA) for 30 min. Finally, visualisation was performed with the Dako Liquid DAB+ Substrate-Chromogen System (Dako, Denmark) and counterstaining with Harris’ haematoxylin.
Each slide was viewed using standard light microscopy (Olympus BX41).
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