Immunocytochemistry was performed as previously described55 (link). The primary antibodies were Anti-Cytomegalovirus Antibody, clone 8B1.2 (1:2,000; Merck Millipore, Billerica, MA, USA), Anti-ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase family member 2) antibody [5H3] (1:1,000; Abcam, Cambridge, MA, USA), Anti-TGF-β1 antibody (1:100; Sigma-Aldrich), anti-αSMA (1:500; Sigma-Aldrich) and anti-fibronectin [IST-9] (1:400; Abcam). Alexa Fluor 488 and 594 secondary antibodies (1:1,000) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). To assess the characteristic changes in hTM and SCE cells after exposure to the conditioned medium, we performed immunocytochemistry using rhodamine phalloidin (7:1,000, Thermo Fisher Scientific) and ZO-1 (1:100; Abcam), followed by Alexa Fluor 488 and 594 secondary antibodies (1:1000; Thermo Fisher Scientific). We further explored whether the changes induced by the conditioned medium could be suppressed by ROCK inhibitors Y27632 (Merck, Kenilworth, NJ, USA) and K115 (KOWA, Nagoya, Japan), as well as Ki16425 (Merck) and SB431542 (Fujifilm, Osaka, JAPAN).
Alexa fluor 488 and 594 secondary antibodies
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 and 594 secondary antibodies are fluorescent-labeled antibodies used in various immunodetection techniques. They are designed to bind to primary antibodies, allowing for the visualization and detection of target proteins or molecules in biological samples. These secondary antibodies exhibit high fluorescence intensity and photostability, making them suitable for applications such as immunofluorescence microscopy, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Anti α sma, by Merck Group (3 mentions)
Anti rhodamine phalloidin, by Thermo Fisher Scientific (2 mentions)
Anti fibronectin, by Abcam (2 mentions)
Anti collagen type 1, by Cell Signaling Technology (2 mentions)
Bodipy, by Thermo Fisher Scientific (2 mentions)
Mitotracker, by Thermo Fisher Scientific (2 mentions)
Anti ucpl antibody, by Merck Group (2 mentions)
Anti mcherry antibody, by Thermo Fisher Scientific (2 mentions)
Y 27632, by Merck Group (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Anti fibronectin ist 9, by Abcam (1 mentions)
Sb431542, by Fujifilm (1 mentions)
Anti enpp2, by Abcam (1 mentions)
Anti tgf β1 antibody, by Merck Group (1 mentions)
Anti s1p, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
A12379, by Thermo Fisher Scientific (1 mentions)
A12381, by Thermo Fisher Scientific (1 mentions)
Chloroquine cq, by Merck Group (1 mentions)
18 protocols using alexa fluor 488 and 594 secondary antibodies
1
Immunocytochemistry for Cell Characterization
2
Immunocytochemistry for Extracellular Matrix and Cytoskeleton
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Eight hours after irradiation, cells were fixed in ice-cold 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 5 min, and blocked in 3% bovine serum albumin for 30 min. Immunocytochemistry was performed as previously described [65 (link)]. The primary antibodies were anti-fibronectin (1:400; Abcam, Cambridge, MA, USA), anti-collagen type I (1:400; Cell Signaling Technology, Danvers, MA, USA), anti-rhodamine phalloidin (7:1000; Thermo Fisher Scientific, Waltham, MA, USA), anti-αSMA (1:500; Sigma-Aldrich Co., LLC St. Louis, MO USA) and anti-SphK (Sphingosine kinase) 2 (1:400; Abcam, Cambridge, MA, USA), anti-EDG5 (S1PR2) (1:200; Santa Cruz Biothechnology, Dallas, TX, USA), and anti-S1P (1:200; Abcam, Cambridge, MA, USA).
Alexa Fluor 488 and 594 secondary antibodies (1:1000) were purchased from Thermo Fisher Scientific. Cells were counter stained with 4′,6-diamidino-2-phenylindole (DAPI). Quantitative results based on immunocytochemistry were quantified with ImageJ 1.49 (NIH Bethesda, MD, USA). Five images of each experiments were taken and the fluorescence intensities were quantified.
Alexa Fluor 488 and 594 secondary antibodies (1:1000) were purchased from Thermo Fisher Scientific. Cells were counter stained with 4′,6-diamidino-2-phenylindole (DAPI). Quantitative results based on immunocytochemistry were quantified with ImageJ 1.49 (NIH Bethesda, MD, USA). Five images of each experiments were taken and the fluorescence intensities were quantified.
3
Immunostaining of Neural Tissue Constructs
4
ATX-Induced Extracellular Matrix Remodeling
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Cells were grown in chamber slides. After serum starvation for 24 h, the cells were treated with 44 µM ATX for 24 h, with or without the ATX inhibitor, HA 130 (1 µM), which was added 30 min before ATX treatment. The cells were fixed in ice-cold 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 5 min, and blocked in 3% bovine serum albumin for 30 min. The primary antibodies were anti-fibronectin (1:400; Abcam, Cambridge, MA, USA), anti-collagen type I (1:400; Cell Signaling Technology, Danvers, MA, USA), anti-rhodamine phalloidin (7:1000; Thermo Fisher Scientific, Waltham, MA, USA), and anti-αSMA (Sigma-Aldrich Co., LLC St. Louis, MO USA, 1:500). Alexa Fluor 488 and 594 secondary antibodies (1:1,000) were purchased from Thermo Fisher Scientific.
5
Purification and Analysis of δ-TT from Annatto
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δ‐TT was purified from a commercial extract of Annatto (Bixa orellana L.) seeds (American River Nutrition Inc, Hadley, MA, USA).28 Primary antibodies against: caspase 3 (9656), cleaved caspase 3 (9664), PARP (9542), BiP (3177), eIF2α (5324), p‐eIF2α (3398), ATF4 (11815), CHOP (2895), IRE1α (3294), PDI (3501) were from Cell Signaling Technology Inc, Boston, MA, USA; SQSTM1/p62 (PA5‐20839) was from Thermo Fisher Scientific, Rodano, Milano, Italy; LC3 (L8918); JNK, p38 and α‐tubulin (T6199) were from Sigma‐Aldrich, Milano, Italy, and cytochrome c (sc‐13560) was from Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA. Horseradish peroxidase‐conjugated secondary antibody and enhanced chemiluminescence reagents were from Cyanagen (Bologna, Italy). Alexa Fluor 488 and 594 secondary antibodies were from Thermo Fisher Scientific.
Z‐VAD‐FMK (the pan‐caspase inhibitor; FMK001) was from R&D System Inc (Minneapolis, MN). The ER stress inhibitors salubrinal (S) and 4‐PBA (4‐phenylbutyrate), the autophagy inhibitors CQ (chloroquine) and Baf (bafilomycin), the translation inhibitor cycloheximide, and analytical grade solvents were from Sigma‐Aldrich; 3‐MA (3‐methyladenine) was from Selleckchem (Munich, Germany).
Z‐VAD‐FMK (the pan‐caspase inhibitor; FMK001) was from R&D System Inc (Minneapolis, MN). The ER stress inhibitors salubrinal (S) and 4‐PBA (4‐phenylbutyrate), the autophagy inhibitors CQ (chloroquine) and Baf (bafilomycin), the translation inhibitor cycloheximide, and analytical grade solvents were from Sigma‐Aldrich; 3‐MA (3‐methyladenine) was from Selleckchem (Munich, Germany).
6
Multimodal Imaging of Mitochondria
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Cells were fixed with 4% paraformaldehyde for 20 min, followed by incubation with blocking buffer (0.3% triton X-100, 5% horse serum in PBS) for 1 h. Primary antibodies were applied at the following dilutions in blocking buffer at 4 degree C overnight: anti-UCPl antibody 1:200 (Sigma, #U6382), anti-mCherry antibody 1:500 (ThermoFisher, #M11217). Alexa-Fluor 488 and 594 secondary antibodies (ThermoFisher) were incubated 1:500 at room temperature. Nuclei were stained with DAPI. BODIPY (Invitrogen, #D3922) was used at 10 μg/mL and incubated for 15 min in PBS. Mitotracker (ThermoFisher #M22426) was used at 100 nM and incubated for 30 min in PBS prior to fixation.
7
Multimodal Imaging of Mitochondria
8
Immunohistochemical Analysis of Brain Tissue
9
Immunohistochemical Analysis of Vascular Proteins
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Paraffinized eyes were cut at a thickness of 7 μm. Sections were deparaffinized with xylene, rehydrated in ethanol, washed in PBS, and placed in an antigen-retrieval solution (Dako A/S, Glostrup, Denmark). Permanganate bleaching was used to eliminate the autofluorescence of the RPE. Sections were then incubated for 1 h with 2% BSA in 0.05% Tween in PBS to block unspecific binding and then incubated overnight at 4 °C with the anti-VAP-1 antibody (1:500; ab115574, Abcam, Madrid, Spain) or anti-collagen IV (1:200; ab6586, Abcam, Madrid, Spain). After washing, sections were incubated with Alexa Fluor 488 and 594 secondary antibodies (Molecular Probes, Invitrogen, Madrid, Spain) at room temperature for 1 h. Slides were coverslipped with a mounting medium containing 4′, 6-diamidino-2-phenylindole (DAPI) for the visualization of cell nuclei (Vector Laboratories, Palex, Sant Cugat del Vallés, Spain). Images were acquired with a confocal scanning microscope (FV1000, Olympus, Hamburg, Germany).
10
Immunohistochemical Analysis of Rat Pancreata
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Rat pancreata were fixed in 10% buffered formalin and paraffin embedded. Sections were stained with hematoxylin and eosin (H&E) or anti-rat CD8a (BioLegend, San Diego, CA) and guinea pig anti-insulin (Dako, Carpinteria, CA). Briefly, the fixed sections were blocked with PBS-AT (2% BSA grade J and 0.5% Triton X-100 in PBS). Sections were incubated with primary antibody at 4°C overnight. After three washes with PBS, the sections were incubated with secondary antibody at room temperature for 1 hour followed by three washes in PBS. Mounting medium, Vectashield with DAPI, (Vector Laboratories, Inc., Burlingame, CA, USA) was added to the sections. Alexa Fluor 488 and 594 secondary antibodies were from Invitrogen (Carlsbad, CA); isotype controls were from BD Bioscience (San Jose, CA). Images were acquired with a Nikon Eclipse Ti series microscope and analyzed with Nikon Elements image analysis software.
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