Eight‐micron‐thick formalin‐fixed paraffin‐embedded (FFPE) tissue sections from the frontal cortex, temporal cortex and hippocampus were cut from the cases listed in Table 1 . Sections were deparaffinised in xylene and rehydrated using graded alcohols. Immunohistochemistry for all antibodies required pressure cooker pre‐treatment for 10 minutes in citrate buffer pH 6.0. Aβ immunohistochemistry also required formic acid pre‐treatment prior to pressure cooking. Endogenous peroxidase activity was blocked in 0.3% H2O2 in methanol for 10 minutes and non‐specific binding blocked with 10% dried milk solution. Tissue sections were incubated with primary antibodies; Aβ (1:100; Dako); AT8 (tau, 1:600; Thermo); Iba1 (microglial, 1:1000; Wako); CD68 (microglial, 1:100, Dako); CR3‐43 (microglial, 1:150, Dako); P2RY12 (microglial, 1:100; Sigma); Glial fibrillary acidic protein (GFAP) (astrocytic, 1:1000 Dako) for 1 h at RT, followed by biotinylated anti‐rabbit IgG (1:200; Dako) or biotinylated anti‐mouse IgG (1:200; Dako) for 30 minutes at RT and Avidin‐Biotin complex (30 minutes; Dako). Colour was developed with di‐aminobenzidine/H202 (30 ). Stained sections were digitised using a Leica SCN400F slide scanner.
Avidin biotin complex
Manufactured by Agilent Technologies
Sourced in Denmark
The Avidin-biotin complex is a widely used tool in various biochemical and biotechnological applications. Avidin, a protein found in egg white, has a high affinity for biotin, a small vitamin molecule. This strong and specific interaction between avidin and biotin forms the core function of the Avidin-biotin complex. The complex is often utilized for the detection, isolation, and immobilization of biomolecules in research and diagnostic settings.
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Lab products found in correlation
Biotinylated anti rabbit igg, by Agilent Technologies (3 mentions)
Biotinylated anti mouse igg, by Agilent Technologies (3 mentions)
Biotinylated secondary antibody, by Agilent Technologies (3 mentions)
Eclipse ni microscope, by Nikon (2 mentions)
Prox 1, by OriGene (2 mentions)
Vegfr3, by R&D Systems (2 mentions)
Goat anti dcx, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti neun, by BD (2 mentions)
P2ry12, by Merck Group (1 mentions)
Scn400f slide scanner, by Leica (1 mentions)
Cr3 43, by Agilent Technologies (1 mentions)
Dm1750m, by Leica (1 mentions)
Lyve 1, by R&D Systems (1 mentions)
Lyve 1, by Abcam (1 mentions)
Normal horse serum, by Agilent Technologies (1 mentions)
Dm2500, by Leica (1 mentions)
3 3 diaminobenzidine substrate kit, by Vector Laboratories (1 mentions)
9 protocols using avidin biotin complex
1
Immunohistochemical Analysis of Neurodegenerative Markers
2
ROS1 Immunohistochemical Staining Protocol
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Tissues were fixed in formalin, sectioned and mounted on poly-l-lysine-coated glass slides. Paraffin sections were deparaffinized, and incubated in antigen retrieval buffer for 2 min at 95°C and then for 10 min at room temperature. The sections were then treated in 3% hydrogen peroxide for 5 min. Non-specific antibody binding was blocked with 5% BSA in TBST. The sections were treated with mouse anti-ROS1 monoclonal antibody (Abcam, Cambridge, UK) overnight at 4°C in PBS, rinsed, and subsequently incubated for 1 h with biotinylated HRP-conjugated goat anti-mouse secondary antibody (Abcam), followed by the avidin-biotin complex (Dako, Copenhagen, Denmark). The sections were developed with DAB, counterstained with hematoxylin, and examined under a microscope (DM1750M; Leica, Solms, Germany) to assess the immunoreactivity.
3
Immunohistochemical Evaluation of CD31
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Routine two-day tissue processing of both the tissues was done using the automated tissue processor; after that the tissues were embedded in paraffin wax; 4-micrometer thick sections of the tissue were cut from the wax block and were placed on two poly-L-lysine coated slides. The routine H&E staining of the slide was done using the H&E staining kit.
The immunohistochemistry of the second slide was done. The sections were deparaffinized in xylol and then they were rehydrated in graded alcohol series. Endogenous block was done using 3% H2O2 in methanol. The sections were washed in distilled water and antigen retrieval was done in E-Z antigen retrieval microwave as per supplier's instructions (in citrate buffer 10 mM, at pH 6). The slides were then incubated at 20°C for 45 min with monoclonal CD31 antibody. The slides were then incubated with anti-mouse biotinylated bridging antibodies (1/200 dilution) for 30 min. Sections were then washed and incubated with standard avidin-biotin complex (Dako) for 30 min. Antibody binding was seen using H2O2 as a substrate and diaminobenzidine as chromogen. The slides were then counterstained using hematoxylin.
The immunohistochemistry of the second slide was done. The sections were deparaffinized in xylol and then they were rehydrated in graded alcohol series. Endogenous block was done using 3% H2O2 in methanol. The sections were washed in distilled water and antigen retrieval was done in E-Z antigen retrieval microwave as per supplier's instructions (in citrate buffer 10 mM, at pH 6). The slides were then incubated at 20°C for 45 min with monoclonal CD31 antibody. The slides were then incubated with anti-mouse biotinylated bridging antibodies (1/200 dilution) for 30 min. Sections were then washed and incubated with standard avidin-biotin complex (Dako) for 30 min. Antibody binding was seen using H2O2 as a substrate and diaminobenzidine as chromogen. The slides were then counterstained using hematoxylin.
4
Immunohistochemical Analysis of Mouse Brain
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6 month-old mouse brains in an AKR background were sectioned and paraffin-embedded [21 (link)]. Sections on slides were deparaffinised in xylene and rehydrated using graded alcohols. Immunohistochemistry for all antibodies required pre-treatment with a pressure cooker for 10 min in citrate buffer pH 6.0. Endogenous peroxidase activity was blocked in 0.3% H2O2 in methanol for 10 min and non-specific binding with 10% dried milk solution. For this specific experiment, tissue sections were incubated with primary antibodies against human proteins that cross-react with mouse; PROX-1 (1:400; Acris), or VEGFR3 (1:40; R&D Systems) for 1 h at RT, followed by biotinylated anti-rabbit IgG (1:200; Dako) or biotinylated anti-mouse IgG (1:200; Dako) for 30 min at RT and Avidin–Biotin complex (30 min; Dako). Colour was developed with di-aminobenzidine/H2O2 [32 (link)].
Images were acquired using a Nikon Eclipse Ni microscope using 10×, 20×, and 40× air objectives and 60× oil objective.
Images were acquired using a Nikon Eclipse Ni microscope using 10×, 20×, and 40× air objectives and 60× oil objective.
5
Immunohistochemical Analysis of Lymphatic Markers
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Formalin-fixed, paraffin-embedded tissue Sections (8 µm) from the frontal and occipital cortices were cut from cases 1–6 listed in Table 1 . Sections were deparaffinised in xylene and rehydrated using graded alcohols. Immunohistochemistry for all antibodies required pre-treatment with a pressure cooker for 10 min in citrate buffer pH 6.0. Endogenous peroxidase activity was blocked in 0.3% H2O2 in methanol for 10 min and non-specific binding with 10% dried milk solution. Tissue sections were incubated with primary antibodies; MRC1 (1:1000; R&D systems), PROX1 (1:400; Acris), LYVE1 (1:50; Abcam), LYVE1(1:200; R&D Systems), PDPN (1:350; Sigma-Aldrich), and VEGFR3 (1:40; R&D Systems) for 1 h at RT, followed by biotinylated anti-rabbit IgG (1:200; Dako) or biotinylated anti-mouse IgG (1:200; Dako) for 30 min at RT and Avidin–Biotin complex (30 min; Dako). Colour was developed with di-aminobenzidine/H2O2 [32 (link)].
Images were acquired using a Nikon Eclipse Ni microscope using 10×, 20×, and 40× air objectives and 60× oil objective.
Images were acquired using a Nikon Eclipse Ni microscope using 10×, 20×, and 40× air objectives and 60× oil objective.
6
Immunohistochemical Analysis of IGFBP-7
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Immunohistochemistry was performed using the method described previously [32 (link)] with minor modification. Four μm sections from paraffin-embedded tissues were deparaffinized and treated with 3% hydrogen peroxidase in methanol. Heat-induced epitope retrieval was achieved by incubation in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven (700 W) by two cycles of 5 min. The sections were then placed in a humidified chamber with 10% normal horse serum (Dako) at room temperature (RT) for 20 min and incubated with primary antibody against IGFBP-7 (1:20, goat polyclonal IgG, sc-6064, Santa Cruz, USA) at RT for 1 h. The sections were rinsed with PBS and incubated with an appropriate dilution of biotinylated anti-goat antibody (Dako) at RT for 1 h, rinsed with PBS, and incubated with avidin-biotin complex (Dako) at RT for 30 min. The substrate chromogen, 3% amino-9-ethylcarbazone (Dako) was developed for 5 to 8 min. The slides were then counterstained with Mayer hematoxylin and photographed. The positive control was sampled from normal oral tongue epithelium tissue known to express IGFBP-7.
7
Immunohistochemical Analysis of Synaptophysin in Cerebellum
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The cerebellum (Fig. 1A) was dissected and fixed in 4% paraformaldehyde (PFA) for 24 h at 4°C. Tissues were processed for paraffin embedding and sagittal sections (5-μm thick) were collected. In brief, after blocking endogenous peroxidase activity, increasing permeability (0.5% Triton X-10, 30 min), and blocking (3% BSA, 1 h at 37°C) sections were exposed to the anti-Syn antibody in 1% BSA (12 h, 4°C, 1:200; Merck, Darmstadt, Germany). Sections were then washed by PBS for three times and incubated with the avidin–biotin complex (DAKO, Glostrup, Denmark) followed by the biotinylated secondary antibody (1:200; 2 h, 37°C; DAKO, Glostrup, Denmark), and finally staining was visualized using the 3,3-diaminobenzidine substrate kit (Vector Laboratories, Burlingame, CA, U.S.A.). Finally, sections were observed under a microscope (Leica DM2500, Germany).
8
Immunofluorescence of Brain Sections
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Immuno-uorescence was conducted according to previous method 15 . Brie y, serial coronal brain sections (5 or 40 µm in thickness) were collected. The sections were rst washed in 0.01 M PBS to remove the cryoprotectant solution. The sections were then incubated with primary antibodies for 12 h at 4 °C, including rabbit anti-NeuN (1:1000, BD, San Jose, CA) or goat anti-DCX (1:200, Santa Cruz Biotechnology, CA, USA). For BrdU staining, all the sections were pretreated with 2 N HCl for 1 h at 37 °C to denature the DNA followed by 0.1 M borax (pH 8.5) treatment for 10 min to neutralize before the regular immunostaining procedure. After washing, the sections were incubated with biotinylated secondary antibody (1:200, Dako, Glostrup, Denmark) (2 h, 37 °C), followed by the avidin-biotin complex (Dako). Finally, sections were analyzed under a Lycra microscope.
9
Immunohistochemistry Protocol for Brain Tissue
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Immunohistochemistry was conducted according to previous method 28 . Brie y, rats were deeply anesthetized with an overdose of iso urane and transcardially perfused with 0.01 M phosphatebuffered saline (PBS, pH 7.4) for 5-10 min, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4) for 15-20 min. Whole brains were removed and post-xed in the same xative for 3-4 d at 4 °C followed by 30% sucrose treatment at 4 °C. Serial coronal brain sections (5 or 40 μm in thickness) were collected. The sections were rst washed in 0.01 M PBS to remove the cryoprotectant solution, and then incubated in 3% H 2 O 2 for 30 min at room temperature to quench endogenous peroxidase, followed by washing with 0.01 M PBS. The sections were then incubated with primary antibodies for 12 h at 4 °C, including rabbit anti-NeuN (1:1000, BD, San Jose, CA) or goat anti-DCX (1:200, Santa Cruz Biotechnology, CA, USA). For BrdU staining, all the sections were pretreated with 2 N HCl for 1 h at 37 °C to denature the DNA followed by 0.1 M borax (pH 8.5) treatment for 10 min to neutralize before the regular immunostaining procedure. After washing, the sections were incubated with biotinylated secondary antibody (1:200, Dako, Glostrup, Denmark) (2 h, 37 °C), followed by the avidin-biotin complex (Dako).
Finally, sections were analyzed under a Lycra microscope.
Finally, sections were analyzed under a Lycra microscope.
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