At day 21 of differentiation, human iPSC-derived cardiomyocytes on 13 mm tissue-culture treated coverslips were rinsed with PBS and treated with 100 nM Mitotracker Far Red (Invitrogen P36970) for 35 min at 37°C. Coverslips were fixed in 4% Paraformaldehyde (Electron Microscopy Sciences, 15710) at 4°C for 15 min, rinsed and stored in PBS. Cells were permeabilized with 0.5% Triton-X100 (Millipore 648466) for 10 min at room temperature, rinsed in PBS, blocked in 3% Bovine-Serum Albumin in PBS-Tween 20 0.1% for 90 min at room temperature, and rinsed in PBS. They were incubated overnight at 4 °C in 1:500 Mouse-anti-α-Actinin (Sigma A7811) and 1:300 Rabbit-anti-Pan-Cadherin (Applied Biosystems AB16505) in 3% BSA in PBS-Tween 0.1% at 4°C overnight. Coverslips were washed on a rocker at room temperature 3X for 5 min in PBS-Tween 0.1%, incubated for 1 hour at room temperature in secondary antibodies of 1:400 Donkey-anti-Mouse-488 (Invitrogen A21202) and 1:400 Donkey-anti-Rabbit-568 (Invitrogen A10042), and washed 3X for 5 min at RT on a rocker in PBS-Tween 0.1%. Cells were then stained in 1 μg/ml DAPI for 10 min at room temperature. Coverslips were mounted in Prolong Diamond (Invitrogen P36970) under a #1.5 coverslip. Imaging was done on a Nikon A1R with ×20 objectives.
Donkey anti rabbit 568
Manufactured by Thermo Fisher Scientific
Sourced in United States
Donkey anti-rabbit 568 is a secondary antibody conjugate used for detecting and visualizing rabbit primary antibodies in various immunoassay techniques. The 568 indicates the fluorescent dye label attached to the antibody, which emits light in the orange-red region of the visible spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Donkey anti mouse 488, by Thermo Fisher Scientific (3 mentions)
Dmi6000b, by Leica (3 mentions)
D9663, by Merck Group (3 mentions)
D9542 1mg, by Merck Group (3 mentions)
Alexa fluor 488 donkey anti mouse, by Thermo Fisher Scientific (3 mentions)
Donkey anti mouse 647, by Thermo Fisher Scientific (3 mentions)
Ab5320, by Merck Group (2 mentions)
Ab39012, by Abcam (2 mentions)
Donkey anti rat 488, by Thermo Fisher Scientific (2 mentions)
Donkey anti rabbit 647, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Prolong diamond, by Thermo Fisher Scientific (1 mentions)
Mouse anti α actinin, by Merck Group (1 mentions)
Mitotracker far red, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit 488, by Thermo Fisher Scientific (1 mentions)
Rat anti gfp, by Nacalai Tesque (1 mentions)
Mouse anti arp3, by Santa Cruz Biotechnology (1 mentions)
Donkey anti goat 647, by Thermo Fisher Scientific (1 mentions)
11 protocols using donkey anti rabbit 568
1
Immunofluorescent Staining of iPSC-derived Cardiomyocytes
2
Immunohistochemical Analysis of TMJ OA
3
Co-localization of p53, Rb, and E7
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The primary antibodies used for co-localization studies included: a rabbit polyclonal antibody to p53 (Santa Cruz Biotechnology, Santa Cruz, CA, sc-6243); a rabbit polyclonal antibody to Rb (Santa Cruz Biotechnology, Santa Cruz, CA, sc-50); and a mouse monoclonal antibody to E7 (Santa Cruz Biotechnology, Santa Cruz, CA, sc-65711). Secondary antibodies used included: donkey anti-mouse 488 and donkey anti-rabbit 568 (obtained from Invitrogen, Waltham, MA). DAPI was used as a nuclear fluorescent stain that binds strongly to A-T rich regions in DNA. Confocal images were obtained with an Olympus Fluoview 100 confocal microscope and companion software FV10-ASW1.6. Images were acquired at a resolution of 1024 × 1024 pixels.
4
Immunohistochemical Analysis of TMJ OA
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TMJ tissue samples collected from control and TMJ OA mice were fixed in 4 % PFA overnight, decalcified with 4.5 % EDTA for 28 d, paraffin-wax embedded, and sectioned at 8 μm. Sections were stained using a Mason’s Trichrome. For immunohistochemistry, sections were dewaxed, treated with 132.2 mmol/L sodium borohydride, permeabilised with methanol and 0.5 % Triton (v/v), blocked in 5 % donkey serum (D9663, Sigma-Aldrich, St. Louis, MO, USA) for 2 h, and incubated with primary antibodies against either MMP-13 (1:200, AB39012, Abcam, Branford, CT, USA) or NG2/CSPG4 ectodomain (1:200, AB5320, Sigma-Millipore, Santa Cruz, CA, USA). All secondary labelling was with Alexa Fluor donkey anti-mouse 488 and donkey anti-rabbit 568 (1:500, Invitrogen, Invitrogen, Carlsbad, CA, USA). Nuclei were labelled with DAPI (D9542-1MG, 1 μg/μL, Sigma-Aldrich). Sections were imaged using an inverted fluorescent microscope using a 10 × objective (DMI6000B, Leica, Buffalo Grove, IL, USA). Laser intensity, gain, and magnification were standardised for all acquisitions. Brightness and contrast settings were standardised for all images during post-processing. Acquisition settings were verified using control stains containing no primary antibody or isotype control (Reed et al., 2022 (link); Yotsuya et al., 2019 (link)). All images are representative of 4 biological replicates for each experimental group.
5
Temporospatial Distribution of FAK and pFAK
6
Immunofluorescent Staining Protocol for Cultured Cells
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Cultured cells were fixed by immersion in 4% (w/v) PFA in PBS overnight at 4 °C and PBS washed before primary antibodies were applied in blocking solution (10% FCS/0.2% Triton X-100 in PBS) overnight at 4 °C. After PBS washing, Alexa Fluor-conjugated secondary antibodies, diluted in blocking solution, were applied overnight at 4 °C. Coverslips were washed thrice and mounted with DAKO fluorescent mounting medium (Sigma). Primary antibodies included: rabbit anti-Pcdh1532 (link) [0.45 mg/ml; 1:200; Figs. 1 and 2 ], rabbit-anti-pan Pcdh15 (HL561429 (link), 1:100; Fig. 8 ), rabbit anti-CD3 Pcdh15 (PB37529 (link), 1:100; Fig. 8 ), rat anti-GFP (Nacalai Tesque, 1:2000), goat-anti-PDGFRa (R&D System; 1:100), mouse-anti-Arp2 (Santa Cruz Biotechnology SC376698; 1:10) and mouse-anti-Arp3 (Santa Cruz Biotechnology SC48344; 1:10). Secondary antibodies included: donkey-anti-rat 488 (Invitrogen; 1:1000), donkey-anti-rabbit 568 (Invitrogen; 1:1000), donkey-anti-rabbit 647 (Invitrogen; 1:1000), donkey-anti-rabbit 488 (Invitrogen; 1:1000), donkey-anti-mouse 647 (Invitrogen; 1:1000) and donkey anti‐goat 647 (Invitrogen; 1:1000). Filamentous (F)-actin was also detected by exposing cells to Alexafluor-568 conjugated phalloidin (Invitrogen; 1:50) and cell nuclei were detected by exposing cells to Hoechst 33342 (Thermo Fisher Scientific; 1:1000).
7
Multicolor Immunofluorescence Staining Protocol
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Free-floating sections were blocked for 2 h at RT in 3% BSA and 0.3% Triton in Phosphate-buffered saline (PBS) and incubated with primary antibodies [rabbit anti-TMEM119; 1:100 (Abcam), mouse anti-CD11c; 1:100 (Abcam), rat anti-CD169; 1:200 (BioLegend) rabbit anti-CD3; 1:100 (Abcam), mouse anti-CD45; 1:500 (BD biosciences), mouse anti-CD68; 1:500 (Abcam), mouse anti-CD317; 1:100 (R&D Systems), mouse anti-CS-56; 1:200 (Abcam), rat anti-GFAP; 1:200 (Thermo Fisher Scientific)], mouse anti-NeuN; 1:100 (Millipore), for 48 h at 4°C. Sections were washed for 3 × 30 min in PBS and incubated for 2 h at RT with secondary antibodies [Donkey anti-mouse 568, 1:500 (Invitrogen); Donkey anti-rat 488, 1:500 (Invitrogen); Donkey anti-rabbit 568, 1:500 (Invitrogen)]; Donkey anti-mouse 647, 1:1,000 (Invitrogen) together with DAPI (1:1,000). After a second round of washing (3 × 30 min in PBS), sections were mounted with Fluorogold prolong antifade mounting medium (Invitrogen). The list of material used has been summarized in Supplementary Table 2 .
8
Pluripotency Marker Immunostaining Protocol
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Cells were fixed with 10% Formalin (Sigma Aldrich) for 15 minutes at room temperature, then washed three times with PBS. They were then incubated with 0.1% Triton X-100 in PBS for 30 minutes with gentle rocking, followed by three additional PBS washes, and incubation with 1% bovine serum albumin (BSA) in PBS for a minimum of 30 minutes at room temperature. Cells were then incubated with primary antibodies prepared in 1% BSA in PBS overnight at 4°C, followed by three PBS washes and incubation with the appropriate secondary antibodies at a 1:100 dilution in 1% BSA in PBS for a minimum of 2 hours at room temperature. Secondary antibodies were then removed, and cells were incubated for 10 minutes at room temperature with 300nM DAPI prepared in PBS. Three PBS washes were repeated, and cells were stored in PBS at 4°C until ready for imaging. For pluripotency confirmation, the following antibodies were used: TRA-1–81 (Cell Signaling Technologies 4745, 1:1000), SSEA4 (Cell Signaling Technologies 4755, 1:1000), TRA-1–60 (Cell Signaling Technologies 4746, 1:1000), Nanog (Cell Signaling Technologies D73G4, 1:400), SOX2 (Cell Signaling Technologies 3579S, 1:400) and OCT4A (Cell Signaling Technologies 2840S, 1:400). Secondary antibodies were AlexaFluors Goat-anti-mouse 488 and Donkey-anti-Rabbit 568, all used at 1:100 and purchased from ThermoFisher Scientific.
9
Microglial-Neuronal Interactions Analysis
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The brains of SAL-injected animals were processed to interrogate microglial-neuronal interactions. Microglia were detected with a rabbit polyclonal antibody against Iba1 (#019-19741; Wako Chemicals). Neurons were detected with a mouse monoclonal antibody against NeuN, clone A60 (MAB377; Millipore). The protocol for all antibodies was similar to that described above, and PBS was used throughout to wash the sections. Sections were blocked with 5% normal donkey serum (NDS) with 0.3% Triton-X in PBS at room temperature for 1 h. Primary antibodies were diluted in a solution of 5% NDS and 0.3% Triton-X in PBS to yield dilutions of 1:1,000 primary antibody for both Iba1 and NeuN. Sections were incubated overnight at 4°C. For fluorescent signal detection, the following Alexa Fluor conjugated secondary antibodies were used: Donkey anti-mouse-488, donkey anti-rabbit-568 (1:500, both purchased from Thermo Fisher Scientific). Sections were incubated in the dark for 2 h at room temperature. Labeled sections were mounted onto glass slides and cover slipped with fluorescent mounting medium (Vector laboratories).
10
Immunofluorescence Imaging of Apoptosis
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Cells were grown on glass cover slips, treated with TNF (100 ng/ml), and fixed for 10 min at room temperature in 4% PFA. Cells were permeabilized and blocked in 0.5% Triton-X, 5% normal donkey serum in PBS. Cleaved caspase 3 antibody (1:400, Cell signaling Technology, #9664) was diluted in blocking solution and incubated overnight at 4 ˚C. Coverslips were washed in PBS and incubated 2 h at room temperature with secondary antibody (Donkey anti-rabbit-568, ThermoFisher Scientific, 1:400), Alexa Fluor 647 Phalloidin (1:1000, Cell signaling technology, #8940) and DAPI. Coverslips were mounted with Prolong Gold (ThermoFisher Scientific) and imaged with a Lunaphore.
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