The protocol used to prepare the tissue sections was the same as Nissl staining. Then, the sections were boiled in sodium citrate antigen repair solution for 20 min, and immersed in 0.5% Triton X-100 for 30 min. Also, cultured microglia and neurons were harvested and fixed. The brain sections or cells were blocked in 5% BSA for 2 h at room temperature. Subsequently, these sections or cells were incubated with the following primary antibodies at 4°C overnight: Iba-1 (1:500 brain sections and 1:1000 cells, ab178846, Abcam, UK), NLRP3 (1:400 brain sections and 1:1000 cells, bs-10021R, Bioss, China), ASC (1:400 brain sections and 1:1000 cells, bs-6741R, Bioss, China), caspase-1 p20 (1:400 brain sections and 1:1000 cells, bs-10442R, Bioss, China), NeuN (1:500, ab177487, Abcam, UK), Active Caspase3 (1:400, bsm-33199M, Bioss, China), and MAP2 (1:200, GTX133109, GeneTex, USA). After washing three times in PBS containing 5% Tween-20, brain sections and cells were incubated in appropriate secondary antibodies at 4°C for 8 h: Goat Anti-Rabbit IgG FITC (1:100, SA00003-2, Proteintech, USA) and Goat Anti-Mouse IgG Alexa Fluor 647 (1:100, ab150115, Abcam, UK), after which cell nuclei were stained with DAPI. Finally, brain sections and cells were imaged under a multichannel fluorescence microscope (Leica, Wetzlar, Germany) and quantified with ImageJ software.
Sa00003 2
Manufactured by Proteintech
Sourced in United States, China
The SA00003-2 is an electrophoresis gel tank system designed for the separation and analysis of DNA, RNA, and protein samples. It provides a controlled environment for the efficient migration of molecules through an agarose or polyacrylamide gel matrix under the influence of an electric field. The core function of this product is to facilitate the separation and visualization of macromolecules for various analytical and preparative applications.
Automatically generated - may contain errors
Lab products found in correlation
Sa00009 1, by Proteintech (5 mentions)
Ab177487, by Abcam (2 mentions)
Sa00003 1, by Proteintech (2 mentions)
Sa00007 1, by Proteintech (2 mentions)
Ab178846, by Abcam (1 mentions)
Alexa fluor 647 goat anti mouse igg, by Abcam (1 mentions)
Bs 10021r, by Bioss Antibodies (1 mentions)
Bs 6741r, by Bioss Antibodies (1 mentions)
Bs 10442r, by Bioss Antibodies (1 mentions)
Bsm 33199m, by Bioss Antibodies (1 mentions)
Goat anti rabbit igg fitc, by Proteintech (1 mentions)
Ab150115, by Abcam (1 mentions)
Cell light edu apollo 567 kit, by RiboBio (1 mentions)
Mab1281, by Merck Group (1 mentions)
Imagej software, by Leica (1 mentions)
Ab18723, by Abcam (1 mentions)
Sig 39320, by Fortrea (1 mentions)
Ctr4000b, by Leica (1 mentions)
Lsm 800, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
28 protocols using sa00003 2
1
NLRP3 Inflammasome Activation Measurement
2
Cellular and Tissue Immunofluorescence Staining
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The cellular immunofluorescence staining for Lamin B1 (1:100, CL594-66095, Proteintech, China) was performed to detect cell senescence. For tissue immunofluorescence staining, after blocking, the frozen sections were incubated with primary antibodies against NeuN (1:1000, ab177487, Abcam, USA), Aβ (mouse anti-Aβ (1:200, SIG-39320, Covance), DCX (1:100, ab18723, Abcam, USA), MAB1281 (1:100, MAB1281, Merck, USA), GFAP (1:500, 16825-1-AP, Proteintech, China) and Iba1 (1:100, 10904-1-AP, Proteintech, China) overnight at 4 °C, and then incubated with Cy3 or FITC-conjugated anti-mouse or ant-rabbit IgG (1:200, AB0122, Abways, China) for 2 h. For EdU/DCX, EdU/NeuN, or EdU/Nestin double immunofluorescence staining, EdU solution (5 mg/kg) was intraperitoneally injected daily for three consecutive days before the mice were sacrificed, according to the instructions of the Cell-Light EdU Apollo567Kit (RiboBio, China). After Apollo staining, the slices were respectively incubated with DCX, NeuN or Nestin antibody (1:1000, 19483-1-AP, Proteintech, USA) overnight at 4 °C and incubated with FITC-conjugated IgG antibody (1:500, SA00003-2, Proteintech, USA) for 2 h. After DAPI (1:100) counterstaining, the sections were examined under a microscope (Leica, German), and the positive cells were analyzed using Image J software (Bethesda, USA).
3
Immunofluorescence Assay for Cartilage Markers
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The treated cells were fixed with formaldehyde for 10mins and incubated in 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the primary anti-COL2A1 (1:50, Santacruz biotechnology, sc-52658) and ACAN (1:200, proteintech, 13880-1-AP) antibody overnight at 4°C. The secondary antibody (green and red) was goat anti rabbit and mouse (proteintech, SA00003-2 and proteintech, SA00009-1) Ig G(H+L) which were used at a dilution of 1 to 50 for 1h. DAPI was used to stain the cell nuclei(blue) and its concentration was 1.43μM. The cells were observed by fluorescence microscope (CTR4000B, Leica). Experiments were repeated three times independently.
4
Immunohistochemical Analysis of Annexin6 and Pik3r2
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After PBS perfusion, the brain tissue of rats were fixed with 4% paraformaldehyde for 24 h, dehydrated with 30% sucrose to the bottom, embedded with OCT glue, quickly frozen with liquid nitrogen and preserved at -80 ℃.The frozen slicer was used to cut into 6 micron slices, fixed with 4% paraformaldehyde for 15 min, washed with PBS for 3 times, 3 min each, and 0.5% Triton for 30 min, washed with PBS for 3 times, 3 min each, and sealed with goat serum for 30 min. rabbit anti-Annexin6 (1:100, Proteintech, 12542–1-AP, USA) and mouse anti-Pik3r2 (1:100, Proteintech, 67644–1-Ig, USA)) primary antibodies were incubated overnight, washed with PBS 3 times for 3 min each, and goat anti-rabbit(1:50, Proteintech, SA00003-1, USA)) and goat anti-mouse(1:50, Proteintech, SA00003-2, USA)) secondary antibodies were incubated at 37° for 1 h, washed with PBS 3 times for 3 min each, stained with DAPI and sealed.The results were observed under a confocal microscope (LSM 800, Zeiss, Germany), and 5 fields at high magnification (200 ×) were randomly selected for observation.
5
Immunostaining of Hippocampal Neuron Proteins
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After treated with CNQX, CI‐HIBO, rapamycin or MHY1485 for 18 hours, hippocampal neurons were fixed with 4% paraformaldehyde for 30 minutes and then treated with 0.25% Triton X‐100 for 15 minutes and blocked in 5% bovine serum albumin for 30 minutes, before incubation with primary antibodies, overnight, in the dark, in a humidified container, at 4℃. The following antibodies were used: rabbit anti‐Parkin polyclonal antibody (1:100, 14060‐1‐AP, Proteintech), rabbit anti‐GluR2 polyclonal antibody (1:100, 11994‐1‐AP, Proteintech), rabbit anti‐mTOR polyclonal antibody (1:100, 20657‐1‐AP, Proteintech), rabbit anti‐Beclin‐1 polyclonal antibody (1:100, ab210498, Proteintech), rabbit anti‐Cyt‐c polyclonal antibody (1:100, 10993‐1‐AP, Proteintech), rabbit anti‐Bax polyclonal antibody (1:100, 50599‐2‐Ig, Proteintech), rabbit anti‐caspase‐9 polyclonal antibody (1:100, 10380‐1‐AP, Proteintech). The hippocampal neurons were then incubated with anti‐rabbit IgG, conjugated to fluorescein isothiocyanate (1:400, SA00003‐2, Proteintech) at 37℃ for 30 minutes and stained with DAPI (1:800, Abcam) at room temperature for 15 minutes. Finally, a laser scanning confocal microscope was used to analyse protein expression.
6
Quantifying CD101 Expression in Tumor-Associated Macrophages
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To estimate the density of the expression level of CD101 on M2-type tumor-associated macrophages, immunofluorescence assay was exploited in our research. Formalin-fixed tissues were paraffin embedded and sliced into 4-μm sections. These sections were installed on slides and managed as previously described (24 (link)). Subsequently, the goat serum containing 0.3% Triton were used for blocking brain slices at room temperature (RT). The primary antibodies, including anti-human CD101 (1:200, 26047-1-AP, Proteintech, Wuhan, China) and anti-human CD163 (1:200, CL594-16646, Proteintech) were used to incubate with slices overnight at 4°C. After being laved in PBS for three times, the slices were incubated with the secondary antibody (1:200, SA00003-2, Proteintech) for 1 h at RT, followed by staining with DAPI (MBD0015, Sigma-Aldrich). Colocation analysis and double-stained cell counts were performed by ImageJ software.
7
Immunohistochemical Analysis of Brown and White Adipose Tissue
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The treated-side SS (n = 5 per group) cryosection slides were fixed in 4% paraformaldehyde for 30 minutes, rinsed in PBS, placed in 0.1 M glycine (diluted in PBS; Fisher Scientific) for 30 minutes, and washed again in PBS. They were then covered with blocking solution (0.2% Triton X-100; 2% bovine serum albumin in PBS) for 1 hour at room temperature. Primary antibodies against UCP1 (an indicator of BAT activation or white fat browning) (diluted 1:100; NB100-2828; Novus Biologicals), tyrosine hydroxylase (TH) (an indicator of activation of sympathetic nerve fibers) (diluted 1:500; 66334-1-Ig; Proteintech), laminin (diluted 1:500; L9393; Sigma-Aldrich), and perilipin (diluted 1:500; abs137082; Absin) were diluted in a block mix and added to the sections for overnight incubation at 4°C. After a PBS rinse, the sections were incubated with a mixture containing FITC-conjugated (diluted 1:500; SA00003-8, SA00003-2; Proteintech) and Cy3-conjugated (diluted 1:500; SA00009-3, SA00009-1; Proteintech) secondary antibodies at room temperature for 120 minutes. After a PBS rinse for 2 × 10 minutes, the slides were covered with DAPI containing antifade mounting medium.
8
Immunofluorescent Imaging of Autophagy in Rat Cerebral Ischemia
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Rats were deeply anesthetized and transcardially perfused with normal saline followed by 4% paraformaldehyde in 0.1 M PBS 24 h post-HI, and then embedded in paraffin after dehydration in graded ethanol. Immunofluorescence was performed on 3.5 μm-thick coronal sections. After hot repairing, the sections were incubated with 10% serum in 0.1 M PBS for 40 mins, and incubated overnight at 4°C with primary antibodies against LC3B (Rabbit polyclonal 1:100; 2775S, Cell Signaling Technology), NeuN (Mouse 1:1,000; 2,742,283, Millipore), or both. Following washing with 0.1 M PBS three times for 5 mins each time, sections were incubated with a FITC-conjugated secondary antibody (Goat anti-rabbit IgG 1:100; SA00003-2, Proteintech) or TRITC-conjugated anti-Mouse IgG (1:100; SA00007-1, Proteintech) at room temperature for 4 hrs. The sections were counterstained with DAPI for 5 mins. Immunofluorescence images were collected using a Nikon Eclipse NI microscope.
9
Immunofluorescence Staining of Cultured Cells
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IEC-6 cells were cultured in standard condition for 12 h before the experiment. Cells were fixed with 4% paraformaldehyde (SenBeiJia, Nanjing, China) for 20 min. Then cells were incubated in 0.2% Triton X-100 (Solarbio) for 5 min. Next, the nonspecific protein binding sites were blocked with 3% bovine serum albumin (BSA)-phosphate-buffered saline ((PBS); ZSGB-BIO, Beijing, China) at 37 °C for 1 h. Then cells were incubated overnight with related primary antibody at 4 °C. After washing with PBS for 15 min, cells were incubated for 1 h at 37 °C with an anti-rabbit secondary antibody (SA00003-2, Proteintech). Cells were then treated with prolongTM gold antifade reagent with DAPI (Invitrogen) and were stored from night. Images were verified using the confocal microscope (FV1000PME, Japan). Colon sections were processed in the same way as above, and the images were verified by fluorescence microscope. Furthermore, the inflammation caused by DSS or TNBS was assessed by hematoxylin and eosin (HE).
10
Immunofluorescence Analysis of Txnip and Trx
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The A549 and PC9 cells were seeded on a cover glass and treated with DMEM, 50 µmol/l C2-ceramide, 50 µmol/l C2-ceramide +100 µmol/l verapamil, and 100 µmol/l verapamil for 24 h. The cells were then fixed in 4% paraformaldehyde for 15 min, permeabilized in 0.5% Triton X-100 for 20 min, and then blocked in bovine serum albumin at room temperature for 30 min. The cells were then incubated with primary antibodies to anti-Txnip (1:200; 18243-1-AP, ProteinTech Group, Inc.) and anti-Trx (1:200; NBP2-52983, Novus Biologicals) from different species at a 1:200 dilution at 4°C overnight. After washing with TBST, then stained with Alexa Fluor 488-labeled goat anti-mouse (1:500; SA00013-1, ProteinTech Group, Inc.) and FITC-labeled goat anti-rabbit secondary antibodies (1:500; SA00003-2, ProteinTech Group, Inc.) were added to the cells and incubated at 37°C in the dark for 1 h. The cell nuclei were counterstained with DAPI (Beyotime Institute of Biotechnology) for 5 min at room temperature. Images were observed using a fluorescent confocal microscope (Olympus IX71, Olympus Corporation).
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