Cells were cultured on coverslips and were fixed with 4% paraformaldehyde for 20–30 min, permeabilized by 0.4% Triton X‐100, and blocked in 1% BSA/1x PBST at room temperature for 1 h. Primary and secondary antibodies were diluted in a ratio of 1:1000 and 1:10,000 with blocking buffer, respectively. The information of primary antibodies used in current study was listed as following: vimentin (ab24525, Abcam), α‐SMA (A2547, Sigma), collagen I (C2456, Sigma), FN1 (ED‐A) (ab6328, Abcam), MMP1 (ab137332, Abcam), MMP2 (GTX104577, GeneTex), MMP8 (GTX61732, GeneTex), MMP9 (ab38898, Abcam), and MMP13 (ab39012, Abcam). For secondary antibodies: anti‐rabbit Dylight 488 (SA5‐10038, Thermo Fisher Scientific, Waltham, MA), anti‐chicken Dylight 550 (SA5‐10071, Thermo Fisher Scientific), anti‐mouse Dylight 633 (35512, Thermo Fisher Scientific), and mouse Dylight 488 (35503, Thermo Fisher Scientific). Samples were immunostained with primary antibody (1:200) at 4°C overnight, and further incubated with secondary antibodies (1:200) conjugated with Dylight at room temperature for 1 h. Nuclei were counterstained with DAPI (1:1000), and the samples were mounted with anti‐fade solution. Fluorescent images were obtained using Leica TCS SP8 confocal microscope and analyzed by ImageJ software to determine the fluorescent intensity.78 , 79 , 80
Ab24525
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab24525 is a laboratory equipment product offered by Abcam. It serves as a core function. No further details can be provided in an unbiased and factual manner while maintaining conciseness.
Automatically generated - may contain errors
Lab products found in correlation
Ab73593, by Abcam (4 mentions)
Ab5694, by Abcam (2 mentions)
Ab34710, by Abcam (2 mentions)
Ab18203, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Ab39012, by Abcam (1 mentions)
Ab137332, by Abcam (1 mentions)
Ab6328, by Abcam (1 mentions)
Gtx104577, by GeneTex (1 mentions)
Anti rabbit dylight 488, by Thermo Fisher Scientific (1 mentions)
Anti mouse dylight 633, by Thermo Fisher Scientific (1 mentions)
C2456, by Merck Group (1 mentions)
A2547, by Merck Group (1 mentions)
Tcs sp8 confocal microscope, by Leica camera (1 mentions)
Ab38898, by Abcam (1 mentions)
Ab189034, by Abcam (1 mentions)
Ab4674, by Abcam (1 mentions)
20 protocols using ab24525
1
Immunofluorescence Staining of Cellular Markers
2
Immunolabeling of Neural Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies used were rabbit polyclonal anti-p16INK4a N-terminal (1:1000) (ab189034; Abcam, Cambridge, UK), chicken polyclonal anti-GFAP (1:2000) (ab4674; Abcam, Cambridge, UK), chicken polyclonal anti-vimentin (1:1000) (ab24525; Abcam, Cambridge, UK), rabbit polyclonal anti-PAX2 (1:200) (71–6000; Invitrogen, Inchinnan, Scotland, UK), and goat polyclonal anti-PAX2 (1:1000) (AF3364; R&D systems, Abingdon, UK).
Secondary antibodies used were (all Alexa Fluor conjugated antibodies from ThermoFisher Scientific, Inchinnan, Scotland, UK) goat antirabbit F(ab’)2 cross-absorbed secondary fluor plus 555 (1:1000) (A48283), goat anti-rabbit 568 (1:1000) (A11036), goat anti-chicken 647 (1:1000) (A21449), donkey anti-goat 647 (1:1000) (A21447), or donkey anti-goat 568 (1:1000) (A11057).
Secondary antibodies used were (all Alexa Fluor conjugated antibodies from ThermoFisher Scientific, Inchinnan, Scotland, UK) goat antirabbit F(ab’)2 cross-absorbed secondary fluor plus 555 (1:1000) (A48283), goat anti-rabbit 568 (1:1000) (A11036), goat anti-chicken 647 (1:1000) (A21449), donkey anti-goat 647 (1:1000) (A21447), or donkey anti-goat 568 (1:1000) (A11057).
3
Immunocytochemical Characterization of hPDLCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hPDLCs at passage 4 were seeded at a density of 1 × 104/well and covered in advance with circular coverslips and incubated for 48 h at 37 °C. Cells were then rinsed and fixed with 4% paraformaldehyde at room temperature. Following a further wash, 0.25% Triton X-100 was added into the 24-well plates, which were incubated at 37 °C for 15 min. After incubated with 1% bovine serum albumin (Gibco, USA) and 22.52 mg/ml glycine in PBS + 0.1% Tween-20, hPDLCs were incubated with anti-vimentin (1:100; ab24525; Abcam, USA) and anti-cytokeratin (1:200; AM06387SU-N; OriGene Technologies, China) primary antibodies overnight at 4 °C. The immunohistochemistry assay kit (SP9001; OriGene Technologies; China) was used for immunocytochemical staining according to the manufacturer’s instructions, and a 3,3′-diaminobenzidine was used to stain positive cells. The cells were examined by an inverted microscope (FSX100; Olympus Corporation, Japan).
4
Immunostaining and Imaging of Viral Constructs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After recording, slices were fixed overnight (about 16 h) in 4% formaldehyde in 0.1 M phosphate buffer and then washed with a phosphate-buffered saline (PBS) solution three times for 15 min each. Slices were blocked in PBS, 5% bovine serum albumin, 0.4% Triton X-100 for 1 h at room temperature. Primary antibodies were incubated for 2 h at room temperature: vimentin (tanycyte marker) 1:500 (Abcam; ab24525) and GFP (for the viral construct) 1:500 (Abcam; ab6556); the same constructs were already characterized for cellular specificity in ref. 21 (link). Slices were then washed three times in PBS and incubated with a fluorescent conjugated secondary antibody (goat anti-rabbit or goat anti-mouse; 1:1,000; Invitrogen). Three final washes in PBS were done and slices were mounted in VectaShield with DAPI (Vector Labs) on a microscope slide. Imaging was performed on a Leica SP5 confocal microscope.
Adv-pTSHR-CatCh and the control (GFP) were made by previously established methods (50 (link)) that involved cloning the 5′ flanking region of the rat TSH receptor gene (51 (link)) and mutagenesis of the channelrhodopsin gene (52 (link)) into a dual-promoter construct (53 (link)).
Adv-pTSHR-CatCh and the control (GFP) were made by previously established methods (50 (link)) that involved cloning the 5′ flanking region of the rat TSH receptor gene (51 (link)) and mutagenesis of the channelrhodopsin gene (52 (link)) into a dual-promoter construct (53 (link)).
5
Immunostaining of Mouse Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All antibodies were obtained from commercial sources. Polyclonal antibodies included anti-αSMA (rabbit anti-mouse, Abcam, ab5694), anti-mesothelin (rabbit anti-mouse, Abbiotec, 250519), anti-vimentin (chicken anti-mouse, Abcam, ab24525), anti-claudin 5 (rabbit anti-mouse, Abcam, ab15106), anti-occludin (rabbit anti-mouse, Abcam, ab168986), anti-WT1 (rabbit anti-mouse, Abcam, ab15249). Secondary antibodies included Texas Red goat anti-rabbit IgG (H+L)(Life Technologies) and FITC goat anti-chicken IgY (H+L)(Life Technologies). Monoclonal antibodies included anti-E-cadherin (rat clone ECCD-2, IgG1, Thermo, 13–1900).
6
Cardiac Cell Immunostaining and Confocal Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At 48 h post-fixation, hearts were sectioned into 200-µm slices. For staining, slices were first incubated for 10 min in blocking solution (3% normal donkey serum (NDS) in PBST), followed by primary antibody staining overnight at 4 °C using the following antibodies: anti-vimentin (ab24525), anti-cardiac troponin I (ab188877) or anti-PGP9.5 (ab108986), purchased from Abcam at 1:200 dilution in blocking solution. Slices were then washed twice in PBST, then stained with secondary antibodies (1 mg ml−1) at 1:500 dilution for 3 h at room temperature using the following: F(ab’)2 anti-chicken 488 (703-546-155) and anti-rabbit 647 (711-606-152) purchased from Jackson ImmunoResearch Laboratories. The slices were then stained with DAPI and washed three times with PBST (30 min per wash). Sections were mounted onto slides and mounted with exPROTOS. Slices were imaged on a confocal microscope (Olympus FV3000).
7
Western Blot Analysis of Extracellular Matrix Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of Collagen 1 a1 (Col1a1), Vimentin and alpha smooth muscle Actin α-SMA was evaluated by western blot. Cells were lysed with lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 1% NP-40, protease inhibitor cocktail (Sigma Aldrich) and phosphatase inhibitor (Roche)), gently vortexed for 20 min at 4 °C and centrifuged for 15 min at 13,200 rpm at 4 °C. Supernatants were quantified by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Twenty micrograms of proteins from cell extracts were loaded and separated in 8% SDS-PAGE. After electrophoresis, the gels were transferred to polyvinylidene difluoride membranes (Millipore), therefore blocked (5% no fat milk in 0.1% Tween 20 TBS) and incubated with the primary Ab (1:1000 in TBST + 2% BSA; overnight at 4 °C or 2 h at room temperature): anti-α-SMA [E184] (ab32575, Abcam), anti-Vimentin (ab24525, Abcam), anti-Col1a1 (ab34710, Abcam) and anti-β-Actin (MAB1501R, Chemicon). After wash, the membranes were incubated with the appropriate HRP-conjugated secondary Ab (1:5000 in TBST + 2% BSA; 2 h at room temperature; anti-mouse A4416 and anti-rabbit A0545, Sigma). The immunoreactivity was detected by ECL reagents (Amersham), acquired with the ChemiDoc imaging system (Image Lab, Bio-Rad).
8
Immunohistochemistry Analysis of Intestinal Markers
9
Multicolor Immunofluorescence Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit polyclonal anti-GFAP (ab7260), rabbit polyclonal anti-actin (ab1801), goat polyclonal anti-Iba1 (ab5076), mouse monoclonal anti-CD68 (ab31630), chicken polyclonal anti-vimentin (ab24525) and a secondary antibody – a donkey anti-goat IgG H&L antibody (conjugated with Alexa Fluor® 488; ab150129), donkey anti-rabbit IgG H&L antibody (conjugated with Alexa Fluor® 488; ab150073), donkey anti-mouse IgG H&L antibody (conjugated with Alexa Fluor® 568; ab175472) and goat anti-rabbit IgG H&L antibody (HRP; ab6721) were acquired from Abcam (Cambridge, UK).
10
Immunofluorescence Staining of Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured cells were fixed using 4% paraformaldehyde (PFA) at room temperature for 20 min. After washing with PBS, cells were incubated with 0.3% Triton X-100 (FUJIFILM Wako) in PBS for 15 min and blocked with PBS containing 10% normal goat serum (NGS) (Thermo Fisher Scientific) for 1 h at room temperature. Cells were then incubated with primary antibodies (chicken anti-vimentin [polyclonal, 1/1000, ab24525, Abcam, Cambridge, UK], rabbit anti-glutamine synthetase [GS] [polyclonal, 1/100, ab73593, Abcam], mouse anti-rhodopsin [Rho] [monoclonal, 1/100, ab5417, Abcam], and rat anti-mouse CD44-PE [monoclonal, 1/100, 1M7, eBioscience, San Diego, CA, USA]) diluted in PBS at 4°C overnight. Next, the cells were incubated with Alexa-conjugated secondary antibodies (Goat anti-Chicken IgY, [H+L], Alexa Fluor™ 488, A-11039, Goat anti-Rabbit IgG [H+L], Alexa Fluor™ 546, A-11035, Goat anti-Mouse IgG [H+L], Alexa Fluor™ 647, A-21235, Thermo Fisher Scientific) diluted in PBS for 1 h at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescence images were acquired using a BZ-X710 confocal microscope (Keyence, Osaka, Japan). For cell counting, at least three fields were selected per dish.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!