The postmortem tissue of the AD cases was provided by BRAIN UK under the ethics approval obtained from the National Research Ethics Committee South Central Hampshire B (REC reference 14/SC/0098). Formalin-fixed paraffin-embedded brain tissue was cut into 5 µm sections on a microtome. Tissue was dewaxed in clearene (Surgipath), and rehydrated in graded alcohols. Endogenous peroxidase activity was quenched by incubation of tissue with 3 % H2O2 in methanol for 10 min, before 3 washes with tris-buffered saline (TBS). Sections were then either incubated for 30 min in 80 % formic acid (Fisher) or in TBS. Sections were blocked for 30 min with medium containing DMEM, FCS BSA. Anti-Aβ antibodies were added at a concentration of 1:1000 in TBS and incubated at 4 °C overnight. Sections were then washed in 3 rinses of TBS before addition of biotinylated rabbit anti-mouse IgG secondary (Abcam, 1:600) and incubated for 1 h. Bound antibody was detected by incubation for 30 min with ABC system (Vector), and washed 3 times in TBS, before developing with diaminobenzidine solution (DAB, Vector). Slides were dehydrated in graded alcohols into clearene, before mounting with pertex™ (Cellpath).
Diaminobenzidine dab solution
Manufactured by Vector Laboratories
Sourced in United Kingdom
Diaminobenzidine (DAB) solution is a substrate used in immunohistochemistry and other biological applications to produce a brown-colored precipitate that indicates the presence of a target antigen. The solution contains diaminobenzidine, a chromogen that undergoes an oxidation reaction in the presence of peroxidase enzymes, resulting in the formation of the colored product.
Automatically generated - may contain errors
Lab products found in correlation
Formic acid, by Thermo Fisher Scientific (1 mentions)
Abc system, by Vector Laboratories (1 mentions)
Pertex, by CellPath (1 mentions)
Avidin peroxidase complex, by Vector Laboratories (1 mentions)
Avidin and biotin blocking solution, by Vector Laboratories (1 mentions)
Bhabp, by Merck Group (1 mentions)
Ab125212, by Abcam (1 mentions)
Abc solution, by Vector Laboratories (1 mentions)
Biotinylated secondary antibody, by Vector Laboratories (1 mentions)
Fitc conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Normal goat serum blocking solution, by Vector Laboratories (1 mentions)
6 protocols using diaminobenzidine dab solution
1
Immunohistochemical detection of Amyloid-beta
2
Hyaluronan Staining Protocol
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Hyaluronan staining was performed as described previously (Cordo Russo et al. 2012) . After deparaffination, to block endogenous peroxidase activity slides were incubated with 3% H 2 O 2 in methanol for 15 min at room temperature (rt). To block non-specific binding sites, sections were incubated with 10% fetal bovine serum in PBS for 30 min at rt, followed by an avidin and biotin blocking solution (Vector, Peterborough, UK). Then, biotinylated-hyaluronan binding protein (bHABP) (Calbiochem, Darmstadt, Germany) was added and incubated overnight. As amplification and revealing system, an avidin-peroxidase complex (Vector, Peterborough, UK) was used. The reaction product was visualized by the addition of a diaminobenzidine solution (DAB) (Vector, Peterborough, UK) followed by counterstaining with Mayer's hematoxylin. To assess non-specific binding of bHABP tissue sections were either stained with bHABP that had been pretreated with 50 U/ml Hyaluronidase form bovine testes (Sigma, USA) in PBS at 4 °C overnight or incubated without the addition of bHABP.
3
Quantifying PSMP Expression in Renal Biopsies
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We used a PSMP polyclonal antibody [3D5, purified and provided by the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Science, Beijing, China (16 (link))] to detect the expression of PSMP in renal biopsies. In brief, the paraffin sections were dewaxed by baking for 90 min at 65°C and deparaffinized in xylene solutions and various alcohol concentrations (100%, 95%, and 75%). The sections were placed in a 3% hydrogen dioxide solution for 8 min and boiled with citrate buffer solution for 15 min. The sections were then blocked with serum for 0.5 h. The PSMP polyclonal antibody, anti-CD68 (1:100; ab125212, Abcam Inc., Cambridge, MA, USA), anti-CD163 (1:50, ZM-0428, Zhongshan Bridge, Beijing, China) were used as primary antibodies, and incubation was performed at 4°C overnight. A biotin-conjugated goat anti-mouse/rabbit IgG antibody (1:100, Zhongshan Bridge, Beijing, China) was used as the secondary antibody, and incubation was performed at 37°C for 30 min. The tissues were colored with diaminobenzidine (DAB) solution (Vector Laboratories, Inc., Burlingame, USA). A semi-quantitative assessment was conducted by Image-Pro Plus software (Image-Pro Plus 6.0, USA), the average positive cells number in at least 5 high-power field (HPF, 40×) or mean integrated optic density (IOD) was calculated.
4
Histological Evaluation of Kidney Morphology
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Paraffin embedded kidney sections were dewaxed and rehydrated with ethanol, and then stained with H&E to evaluate kidney morphology, and collagen content was determined by Sirius Red staining [25 (link),26 (link)].
For immunohistochemistry staining, antigens were retrieved using citrate buffer (pH 6). Quenching of endogenous peroxidase activity was performed using 3% hydrogen peroxide for 10 min. The kidney sections were blocked with blocking solution for 1 h and then incubated with the primary antibody overnight at 4 °C. The following day, the kidney sections were incubated with the appropriate HRP-conjugated secondary antibody followed by incubation with ABC solution (Vector Laboratories, Newark, NJ, USA). The immunoreactivities were detected by diaminobenzidine (DAB) solution (Vector Laboratories). Finally, the sections were counterstained with hematoxylin. The immunostaining was examined by a microscope equipped with a digital camera [27 (link),28 (link)]. The number of positive cells were qualified using Image Pro-Plus in a blinded manner.
For immunohistochemistry staining, antigens were retrieved using citrate buffer (pH 6). Quenching of endogenous peroxidase activity was performed using 3% hydrogen peroxide for 10 min. The kidney sections were blocked with blocking solution for 1 h and then incubated with the primary antibody overnight at 4 °C. The following day, the kidney sections were incubated with the appropriate HRP-conjugated secondary antibody followed by incubation with ABC solution (Vector Laboratories, Newark, NJ, USA). The immunoreactivities were detected by diaminobenzidine (DAB) solution (Vector Laboratories). Finally, the sections were counterstained with hematoxylin. The immunostaining was examined by a microscope equipped with a digital camera [27 (link),28 (link)]. The number of positive cells were qualified using Image Pro-Plus in a blinded manner.
5
Immunohistochemical Analysis of Tissue Markers
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Sections were deparaffinized, and the antigen retrieval was performed for 20 min at 98 °C in citrate buffer (10 mM, pH = 6.0). Sections were then blocked in 2.5% Normal Goat Serum Blocking Solution (Vector) for 1 h at room temperature, followed by incubation with primary antibodies (α-SMA, 1:500; Col-1, 1:400; Fibronectin, 1:200; CD31, 1:200; PCNA, 1:200; Arg1, 1:200; CD206, 1:900; iNOS, 1:50; TGFβ1, 1:100; F4/80, 1:200; LIGHT, 1:200) overnight at 4℃. For IHC, the biotinylated secondary antibody (Vector) was applied, and the signal detection was performed using diaminobenzidine (DAB) solution (Vector). For immunofluorescence, sections were incubated with FITC-conjugated secondary antibody (Invitrogen) for 1 h at room temperature. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen).
6
Immunohistochemical Detection of RHAMM and CD44
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After deparaffination, a heat retrieval antigen step was performed. Blocking endogenous peroxidase activity and non-specific binding sites steps were carried out as described above. Slides were then incubated with 1:50 dil of RHAMM polyclonal antibody (E-19, sc-16170, Santa Cruz Biotechnology, Santa Cruz, USA) or 1:25 dil of CD44 monoclonal antibody (IM7, ATCC, USA) overnight at 4 °C. Slides were washed and incubated with anti-goat IgG-HRP and anti-rat F(ab)'2-HRP (Santa Cruz Biotechnology, Santa Cruz, USA) 2 h and overnight at 4 °C at rt, respectively. Slides were washed again and the reaction product was visualized by adding a diaminobenzidine (DAB) solution (Vector, Peterborough, UK) followed by counterstaining with Mayer's hematoxylin. For negative controls, no primary antibody was added. For positive controls, prostate and colorectal cancer tissues were used for RHAMM and CD44, respectively (Gust et al. 2009; Lugli et al. 2010) .
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