A 4 μm thick tissue sections were cut from the paraffin-embedded tissues. After dewaxing and rehydration, 3% hydrogen peroxide was used to block endogenous peroxidase. Non-specific binding was blocked with normal goat serum for 1 h at 37°C. Sections were incubated with anti-E-cadherin antibody (1:200, 24E10, CST) overnight at 4°C. The slides were incubated with Solution I (PV9001, Zsbio, China) for 40 min at 37° C and then incubated with Solution II (PV9001, Zsbio, China) for 40 min at 37°C according to the manufacturer's instruction. After washing with PBS, it was developed with DAB (CW0125, CWBIO, China). The slides were counterstained with Mayer's hematoxylin, washed, dehydrated, and the coverslips were mounted with neutral glue.
3 3 diaminobenzidine (dab)
Manufactured by CWBIO
Sourced in China
DAB is a chromogenic substrate used to visualize the presence of target proteins in immunohistochemistry (IHC) and immunocytochemistry (ICC) applications. It produces a brown reaction product at the site of the target protein, allowing for easy detection and analysis.
Automatically generated - may contain errors
4 protocols using 3 3 diaminobenzidine (dab)
1
Immunohistochemical Analysis of E-cadherin
2
Tissue and Cell Immunostaining Protocol
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For tissue staining, dorsal skin were fixed with 4% paraformaldehyde, dehydrated in graded ethanol series and embedded in paraffin. Sections of 5 µm thickness were prepared by Leica RM2255 rotary microtome and used for immunostaining after deparaffinization. For cell staining, Ptch−/− MEF cells cultured on cover-slips were fixed with 4% paraformaldehyde, blocked with 5% BSA for 1 h and incubated overnight with primary antibody. The following antibodies were used for immunostaining: Smoothened (Santa Cruz, sc-166685, 1:100); ARL 13B (Proteintech, 17711-1-AP, 1:100); Gli1 (Proteintech, 66905–1-Ig, 1:200). The secondary antibodies conjugated with Alexa Fluor 488 or 568 were purchased from Invitrogen, those conjugated with horse radish peroxidase (HRP) using DAB as chromogen from Cwbio. Sections were photographed by LSM710 Zeiss confocal microscope and analyzed with Image J software.
3
Immunohistochemical Localization of Tyrosine Hydroxylase in Striatum
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DAB immunostaining was performed on striatum50 (link). We immersed sections in 3% H2O2 for 30 min at room temperature. Next, 30-mm sections were incubated with anti-TH antibody overnight at 4 ℃. Then used the Vectastain ABC kit followed incubation with biotinylated secondary antibody. The peroxidase reaction product was detected using 3ʹ3ʹ-diaminobenzidine-tetrahydrochloride (DAB; CWBIO). Washed with PBS three times, sections were dehydrated in a graded series of ethanol and immersed in xylene.
4
ICAM-1 Protein Expression Analysis
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RMMVECs were washed one time with pre-cold 1 × DPBS. 200 μL of RIPA lysis buffer was added into each well and incubated at 4 °C for 3-5 min. The cellular lysates were transferred into a 1.5 mL centrifuge tube and gently shaked once every 5–6 min on an oscillator for total 4 times. The cellular lysates were centrifuged at 10000 g for 3 min and the supernatant was taken to run SDS-PAGE; the separated proteins were transferred to PVDF membrane, and blocked with blocking solution (Cwbiotech; Beijing, China) on shaker table for 1 h at room temperature. After being washed with 1 × TBST (Cwbiotech; Beijing, China) three times, the PVDF membrane was incubated with goat anti-rat ICAM-1 antibody (R&D Systems; Minneapolis, USA) (1:2000) at 4 °C the overnight. The membrane was washed with TBST for three times and incubated with HRP-conjugated rabbit anti-goat IgG (1:5000) at 37 °C for 1 h. After washing, 1 ml DAB (Cwbiotech, Beijing, China) was used to develop color and the stripe gradation were analyzed, we use the PBS as blank control.
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