LSK-CD34− cells were stained with cell surface antibodies prior to cell sorting on glass slides and microscopy. For colocalization of LR and Tgfbr1, we used AF555-conjugated CTB (C-34776, Thermo Fisher Scientific, 1 µg/mL) and anti-Tgfbr1 (PA5-38718, Thermo Fisher Scientific, dilution ratio 1:100) with secondary anti-rabbit AF488 (A27034, Thermo Fisher Scientific, dilution ratio 1:1000) antibodies. For the phospho-Smad2/3 microscopy, cells were stained with the cell surface markers (LSK-CD34−) and AF555-conjugated CTB (C-34776, Thermo Fisher Scientific, 1 µg/mL), prior to fixation/permeabilization using BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (BD Biosciences), then we stained with an anti-phospho-Smad2(S465/S467) antibody (AB3849, Merck, dilution ratio 1:250) and with secondary anti-rabbit AF488 (A27034, Thermo Fisher Scientific, dilution ratio 1:1000) antibody. Cells were sorted on glass slides and fixed with ProLong Gold Antifade reagent containing DAPI (P36931, Thermo Fisher Scientific). Images were acquired with an Axio Imager M2 (Zeiss) coupled with an Apotome.2 (×63 objectives) and processed for colocalization studies (Fiji, NIH software). Further information regarding immunofluorescence assays are described in the supplementary Methods section.
A27034
Manufactured by Thermo Fisher Scientific
Sourced in United States
The A27034 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed to perform specific functions within a laboratory setting. The core function of this product is to facilitate laboratory operations, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Ab34710, by Abcam (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Ab75766, by Abcam (1 mentions)
Fortessa flow cytometer, by FlowJo (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Triton x 100, by Carl Roth (1 mentions)
Ab45932, by Abcam (1 mentions)
Ab25088, by Abcam (1 mentions)
Ab19026, by Merck Group (1 mentions)
Prolong diamant antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
A11003, by Thermo Fisher Scientific (1 mentions)
Bz x710 all in one fluorescence microscope, by Keyence (1 mentions)
Bz x viewer v1, by Keyence (1 mentions)
Ab58552, by Abcam (1 mentions)
Wheat germ agglutinin (wga), by Thermo Fisher Scientific (1 mentions)
Ma3 916, by Thermo Fisher Scientific (1 mentions)
A21121, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Electron Microscopy Sciences (1 mentions)
14 protocols using a27034
1
Localization of LR and TGF-β Signaling in LSK-CD34- Cells
2
Visualizing and Quantifying Ciliary Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded at a density of 100,000 cells/well in chamber slides (Ibidi, cat. no. 80822). After 24h, cells were serum-starved for 48h, fixed for 10 min. in 10% formalin, rinsed with PBS and permeabilized with 0.5% Triton X-100 in PBS for 5 min., blocked with 1% fish gelatin in PBS, stained with antibodies to detect Arl13 (ciliary marker; ProteinTech cat. no. 17711-1-AP; secondary antibody: Invitrogen A27034 labeled with Alexa Fluor 488), γ-tubulin (ciliary basal body marker; SIGMA cat. no. T5192; secondary antibody Invitrogen A27034 labeled with Alexa Fluor 488) or eGFP (Aves cat. no. GFP-1020; secondary antibody: Invitrogen A21449 labeled with Alexa Fluor 647), mounted (Invitrogen ProLong cat. no. P36981) and imaged using Zeiss LSM700 confocal microscope. Freshly dissected embryos or kidneys were frozen in OCT (Tissue-Tek; Sakura Finetek USA) and frozen sections were processed as above; or embedded in paraffin, sectioned, and stained with trichrome staining or used for immunohistochemistry. Images were visualized using Imaris (Bitplane). Cystic index was calculated by delineating kidneys in trichrome stained slides using the magnetic lasso feature in Photoshop, and measuring total kidney area, thresholding cystic area and measuring total kidney area and cystic area using Fiji [51 (link)].
3
Quantification of Prohibitin Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NSC-34, SK-N-SH (8 × 106 cells) and RD (4 × 106 cells) cells were seeded onto 6-well plate overnight. The cells were dislodged by incubating them in 10 mM EDTA/PBS in 4°C for 10 mins and spun down to collect the cell pellet. Cells were then blocked with either human or murine Fc-blocker (564200 or 553142, BD Pharmigen; 1:400) for 30 min at RT, and subsequently stained with anti-PHB antibody (AB75766, Abcam; 1:200) and/or anti-rabbit AF488 antibody (A27034, ThermoScientific; 1:500) for 30 min at 4°C. Stained cells were then fixed with 4% PFA. Flow cytometry analysis was carried out using the Becton-Dickinson Fortessa flow cytometer and analysed using FlowJo v10.
4
Immunocytochemical Staining of IRF9
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunocytochemical staining, cells were seeded in eight-well glass chamber slides (Sarstedt, Nürnbrecht, Germany) and grown to 70% confluence. Cells were then washed with PBS at pH 7.4, fixed with 4% paraformaldehyde (Sigma-Aldrich, 158127), and treated with 0.3% Triton X-100 (Carl Roth, 3051.3). After blocking with 1% BSA for 1 h, cells were incubated with IRF9 antibody (1:100; SCBT, sc-10793) was applied for 90 min, washed thoroughly, and incubated with goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (1:1000; Thermo Fisher, A27034, Thermo Fisher Scientific, Waltham, MA, USA) was applied for 1 h. The slides were visualized under a confocal microscope (Zeiss LSM 710, Carl Zeiss, Oberkochen, Germany) using Zen 2011 software.
5
Endogenous Localization of ADAM10 and CX3CL1 in Cardiac Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze the endogenous localization of ADAM10 (1:100, AB19026, Millipore) and CX3CL1 (1:100, ab25088, Abcam), heart tissue sections (mid-ventricular short axis sections, 4% PFA fixed, paraffin embedded, 3 µm) and indicated cells were stained with specific antibodies and counterstained with CD31 (1:100, 553370, BD Biosciences), CD45 (1:100, 555480, BD Biosciences), cTnI (1:100, MAB3150, Millipore), cTnT (1:100, ab45932, Abcam) or Vimentin (1:100, 550513, BD Biosciences) where indicated. Alexa Fluor 488-coupled goat anti-rabbit (1:200, A27034, ThermoFisher Scientific), Alexa Fluor 546-couppled goat anti-mouse (1:200, A-11003, ThermoFisher Scientific) or CF 594-coupled goat anti-rat (1:200, SAB4600323, Sigma-Aldrich) and ProLong Diamant Antifade Mountant with DAPI (P36962, ThermoFisher Scientific) were used as secondary antibodies and for mounting, respectively. Image acquisition was performed using a Keyence BZ-X710 All-in-One Fluorescence Microscope (Keyence) with the BZ-X Viewer v1.03.00.05 software (Keyence). The cellular distribution of ADAM10 and CX3CL1 was assessed using CellProfiler 446 . Cardiac-TnT, CD31, CD45 and Vimentin positive cells were analyzed for ADAM10 expression. For each mouse at least 1000 cells/staining were analyzed. Cardiomyocyte cross-sectional diameter was determined using Fiji. One hundred cells/mouse were analyzed.
6
Cryopreserved Cardiac Tissue Imaging
7
Immunohistochemical Analysis of Human Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry was performed as previously published [33 (link)]. In brief, human brain sections were dewaxed in xylene and rehydrated in ethanol. Antigen retrieval was performed in citrate buffer. Following blocking, samples were incubated with primary antibodies: rat anti-lipocalin-2 (1:500, R&D Systems, MAB1757), mouse anti-IBA1 (1:300, Invitrogen, MA5-27726) or rabbit anti-GFAP (1:250 Millipore, MAB144P) overnight at 4 °C. Samples were washed and incubated with secondary antibodies: goat anti-rat Alexa Fluor 647 (1:500, Thermo Fisher Scientific, A21247), goat anti-mouse Alexa Fluor 647 (1:300, Thermo Fisher Scientific, A28181) or goat anti-rabbit Alexa Fluor 488 (1:500, Thermo Fisher, A27034) for 1 h at RT. Nuclei were labeled with DAPI (1 µg/mL, 5 min, Molecular Probes, Ottawa, ON, Canada) and samples were mounted on microscope slides using Dako mounting medium (Dako, Burlington, ON, Canada). Samples were imaged using a fluorescence microscope. The samples were harvested under a protocol approved by the Montreal Neurological Hospital’s research ethics board (NEU-10-066). Consent was given by all patients and controls (aged 55–76). Tissues were from the cerebral cortex.
8
Laminin Immunostaining in Larvae
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Larvae were euthanized and placed in a 4% formaldehyde solution O/N at 4°C. Then the larvae were washed twice with 1× PBS, permeabilized in acetone for 15 minutes at −20°C, and incubated in PBDT blocking solution (PBS, 1% BSA, 1% DMSO, and 0.5% Triton-X100) O/N. The larvae were then incubated with anti-α-laminin at a 1:25 dilution (Sigma-Aldrich, L9393) O/N at 4°C. The following day, the samples were washed and incubated with goat anti-rabbit IgG Alexa Flour 488 using a 1:250 dilution (Thermo Fisher Scientific, A27034) and 1 µM/mL DAPI O/N at 4°C. The samples were then washed for 30 minutes, three times with a washing solution (1× PBS, 0.1% Tween-20, and 0.1% Triton X-100). Some larvae were stained with phalloidin (300 units/mL) and 1 µM/mL DAPI. Samples were imaged on a confocal microscope (Olympus Fluoview FV3000 confocal microscope at 60× magnification), and images were transferred to cellSENS version 2.3 for deconvolution with five iterations.
9
Immunofluorescence Staining of Micromass Cultures
10
Immunofluorescence Staining of GFP-Labeled Tissue
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!