GC tissue specimens were cut into 5-μm-thick sections. The sections were blocked with normal goat serum and immunostained with primary antibodies to active YAP, CDX2 and Rab27a overnight at 4 ºC, and then with biotin-labeled secondary antibody goat anti-rabbit or goat anti-mouse (BM3894 or BM3895, 1: 500, Boster). Next, the sections were incubated with 50 μL streptomyces anti-biotin–peroxidase solution at room temperature for 10 min and developed with DAB. Following counterstaining, dehydration, clearing and mounting, the sections were observed with a microscope. Primary antibody was substituted by PBS. Positive cells were brown-yellow and the sum of the integrated optical density (IOD) was analyzed using Image-ProPlus6.0 [20 (link)].
Bm3894
Manufactured by Boster Bio
Sourced in China
BM3894 is a laboratory equipment product manufactured by Boster Bio. It is designed for general laboratory use. The core function of BM3894 is to perform specific tasks required in a research or testing environment.
Automatically generated - may contain errors
Lab products found in correlation
Bm3895, by Boster Bio (2 mentions)
Ba2913, by Boster Bio (2 mentions)
D085075, by Bio-Rad (2 mentions)
Ba0056, by Boster Bio (2 mentions)
Pb0517, by Boster Bio (2 mentions)
Frozen slicer, by Leica (2 mentions)
Ab201060, by Abcam (2 mentions)
Zen 2, by Olympus (2 mentions)
Ab150073, by Abcam (2 mentions)
Af5457, by Affinity Biosciences (1 mentions)
Af5147, by Affinity Biosciences (1 mentions)
Af0227, by Affinity Biosciences (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Orb471854, by Biorbyt (1 mentions)
Ab210703, by Abcam (1 mentions)
Ab178846, by Abcam (1 mentions)
Ab7260, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Ab175354, by Abcam (1 mentions)
8 protocols using bm3894
1
Immunohistochemical Analysis of GC Tissues
2
Proteomic Analysis of Urethral Tissue
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Based on a previously reported protocol [1 (link)], the dorsal neurovascular bundle and buck fascia were removed from the TA and urethra. The tissues and fibroblasts in each group were lysed in RIPA buffer (R0010, Solarbio, China). Twelve per cent SDS-PAGE was used to separate proteins (30 μg), and the separated proteins were transferred to PVDF membranes (EMD Millipore). Appropriate primary antibodies diluted with 5% BSA were incubated with the membranes overnight at 4 °C. The primary antibodies consisted of collagen 3 (1:800, AF5457, Affinity, China), Smad7 (1: 800, AF5147, Affinity, China), elastase-2B (1: 800, orb471854, Biorbyt, China), osteopontin (1: 800, AF0227, Affinity, China) and GAPDH (1:1000, BA2913, Boster, China). After 3 washes with TBS-0.01% Tween 20, the HRP-Affinipure goat anti-rabbit IgG (H + L) (1:500, BM3894, Boster, China) was incultured with the membranes for 2 h at 25 °C. After washing, the signals were visualized using enhanced chemiluminescence reagent (D085075, Bio-Rad, China).
3
Immunofluorescence Analysis of Mouse Brain
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After the behavioral experiment, three mice were randomly selected from each group for the immunofluorescence, they were perfused with 0.9% NaCl and 4% paraformaldehyde under isoflurane anesthesia. Mouse brain tissue was fixed in a 4% formaldehyde solution and then dehydrated in a gradient of 20% and 30% sucrose solution. The processed brain tissue is processed into slices with a thickness of 25 microns by a frozen slicer (Leica Microsystems, Wetzlar, Germany). The tissue sections were sequentially reacted with 0.3% TritonX-100, 10% goat serum, target primary and secondary antibodies to complete immunofluorescence staining. The main antibodies are: Anti-ß-amyloid (ab201060, Abcam, 1:200); Anti-GFAP (BA0056, BOSTER, 1:100); Anti-IBA1 (PB0517, BOSTER, 1:100); Goat Anti-Rabbit IgG (BM3894, BOSTER, 1:1000); Dnk pAb to Rb IgE (ab150073, Abcam, 1:1000). Finally the images were imaged using a fluorescent microscope and analysed using ZEN 2.0 software (Olympus, Japan).
4
Immunofluorescence Analysis of Mouse Brain
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After the behavioral experiment, three mice were randomly selected from each group for the immunofluorescence, they were perfused with 0.9% NaCl and 4% paraformaldehyde under isoflurane anesthesia. Mouse brain tissue was fixed in a 4% formaldehyde solution and then dehydrated in a gradient of 20% and 30% sucrose solution. The processed brain tissue is processed into slices with a thickness of 25 microns by a frozen slicer (Leica Microsystems, Wetzlar, Germany). The tissue sections were sequentially reacted with 0.3% TritonX-100, 10% goat serum, target primary and secondary antibodies to complete immunofluorescence staining. The main antibodies are: Anti-ß-amyloid (ab201060, Abcam, 1:200); Anti-GFAP (BA0056, BOSTER, 1:100); Anti-IBA1 (PB0517, BOSTER, 1:100); Goat Anti-Rabbit IgG (BM3894, BOSTER, 1:1000); Dnk pAb to Rb IgE (ab150073, Abcam, 1:1000). Finally the images were imaged using a fluorescent microscope and analysed using ZEN 2.0 software (Olympus, Japan).
5
Immunohistochemical Analysis of Tumor Samples
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The tumor tissues were made into paraffin blocks and cut into sections (3 μm thick). The sections were dewaxed with xylene and hydrated in different volume fractions of ethanol (100%, 95%, 80%, 70%). After washing with phosphate buffered saline (PBS), the sections were submerged in sodium citrate antigen repair solution and heated to boiling for 15 min. After washing, the sections were incubated with 3% H2O2 for 10 min. Diluted primary CD200 rabbit antibody (#23451S, 1:200, Cell Signaling Technology, Shanghai, China), CD200R rabbit-antibody (1:400, #DF4715, Affinity, China), Ki-67 rabbit-antibody (1:400, #9027, Cell Signaling Technology) and CTSK rabbit antibody (1:400, #DF6614, Affinity, China) were incubated with the section at 4 °C for overnight. After rewarming and washing, the sections were incubated with secondary antibody (1:800, BM3894, Boster, China) at 37 °C for 20 min. After washing, the sections were incubated with diaminobenzidine solution (50 μL) for 3 min, and stained with hematoxylin dye solution for 60 s. Finally, the sections were soaked in different volume fractions of ethanol (70%, 80%, 90%, 95%, 100%) for 5 min and xylene for 15 min, 3 times. Neutral gum was used to seal the slides. The results were observed under an optical microscope.
6
Immunohistochemical Analysis of FBXW7
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Sections of lungs and tails were analyzed by immunohistochemistry assay as previously described (Wang et al., 2012 (link)). In this assay, the primary antibody is FBXW7 antibody from rabbit (dilution of 1:400, Abcam) or mouse (dilution of 1:200, Santa Cruz), and the secondary antibody is the HRP-conjugated goat anti-rabbit IgG (dilution of 1:200) (BM3894; Boster Biological Technology co. ltd).
7
Quantifying Alzheimer's Biomarkers in Mice
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After all the behavioral tests were finished, the animals were anesthetized with pentobarbital sodium (50 mg/kg) before being decapitated for the brain. The hippocampi and the cortexes in the two hemispheres of the brain were separated on ice and then used for ELISA detection and the Western Blot experiment, respectively. The levels of tau (ab210703, 1 : 1000), p-tau (ab210703, 1 : 1000), GFAP (ab7260, 1 : 5000), Iba-1 (ab178846, 1 : 1000), and ß-actin (ab6276, 1 : 5000) (all the antibodies were from Abcam company) in the hippocampus and the cortex were determined, respectively. The secondary antibodies were from Boster (bm3895 and bm3894). The homogenates from hippocampus and cortex samples of mice were separately added along with 30 mg total DNA to 10% SDS-PAGE gel for separation. They then were electrophorized under constant voltage, transferred onto a membrane, and incubated with specific antibodies for immunoblotting assays using the ECL immunoblotting analysis system. Each gel contains molecular weight markers (10–170 kDa). The band data obtained were processed with ImageJ (NIH) and the background mean and its standard error were calculated.
8
Western Blot Analysis of Signaling Proteins
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Proteins (30 μg) were separated with 12% SDS-PAGE (Bio-Rad, China) and transferred to PVDF membranes. After blocking with 5% milk, the membranes were incubated with appropriate primary antibodies diluted with 5% bovine serum albumin (BSA) for overnight at 4°C. The primary antibodies consisted of MAPK 1 (AMPK1 or ERK1, 1: 2000, ab109282, Abcam), MAPK14 (another name p38α MAPK, 1:2000, ab170099, Abcam), AKT serine/threonine kinase 2 (AKT2, 1: 1000, ab175354, Abcam), mitogenactivated protein kinase kinase 2 (MAP2K2 or MEK2, 1: 10000, ab32517, Abcam) and GAPDH (1:1000, BA2913, Boster, China). After washing with TBS-0.01% Tween 20 for 3 times, the second antibody HRP-Affinipure Goat anti-Rabbit lgG (H+L) (1:500, BM3894, Boster, China) was cultured with the membranes for 2 h at 25°C. After washing, the signals were visualized by enhanced chemiluminescence reagent (D085075, Bio-Rad, China). The intensity of each band was analyzed using Image J software.
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