Immunocytochemistry was performed as follows: AR42J cells were grown and differentiated on cover slips and were subject to treatments as indicated. After fixation with 4% paraformaldehyde/PBS for 20 min, the cells were permeabilized with Fmbilztzin solution (2.5% Triton/PBS) and incubated in blocking solution (1% Triton, 3% iNGS/PBS) for 1 h followed by overnight incubation with primary rabbit antibodies against XBP1 (Abcam, Israel), detected using goat anti rabbit-IgG coupled to Alexa555 (Abcam, Israel). Nuclear labeling was performed with DAPI (Sigma, Israel). Immunofluorescence were visualized with a LSM 700 Zeiss confocal microscope.
Alexa 555
Manufactured by Abcam
Sourced in United Kingdom
Alexa 555 is a fluorescent dye used as a labeling reagent. It has an excitation maximum at 555 nm and an emission maximum at 565 nm, making it suitable for various fluorescence-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488, by Abcam (3 mentions)
Alexa 647, by Abcam (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Goat anti rabbit igg, by Abcam (1 mentions)
Mab to hcv core antigen, by Abcam (1 mentions)
Lsm 980 confocal microscope, by Zeiss (1 mentions)
Ep1536y, by Abcam (1 mentions)
Apotome 2 microscope, by Zeiss (1 mentions)
Lsm 880 confocal laser scanning microscope, by Zeiss (1 mentions)
Ldh release, by Merck Group (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Abcam (1 mentions)
Normal goat serum (ngs), by Abcam (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Cellinsight cx7 high content screening platform, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342 dye, by Thermo Fisher Scientific (1 mentions)
α sma, by Abcam (1 mentions)
10 protocols using alexa 555
1
Immunocytochemistry of XBP1 in AR42J Cells
2
Visualizing TfR1 and HCV Core
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Cells in chamber slide wells were fixed with paraformaldehyde and stained as follows. Primary antibodies against TfR1 (eBioscience, San Diego, CA, USA) and HCV core (mAb to HCV core Antigen, Abcam, Cambridge, MA, USA) were incubated at a 1:2000 dilution overnight at 4 °C. Conjugated secondary antibodies (anti-mouse Alexa-555, Abcam) were incubated at a 1:1000 dilution for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured via confocal microscopy (BX51-DP70 system, Olympus, Tokyo, Japan) and analyzed using Olympus DP Controller and Management software.
3
Evaluating α-Synuclein Expression in MSA and LBD
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Ten MSA and ten LBD cases were provided through the OADC neuropathology core. Standard histological methods were used to evaluate aSyn expression in the frontal gyrus, hippocampus, amygdala, and midbrain for all cases, and in the putamen and pons in MSA cases. Briefly, formalin-fixed, paraffin-embedded (FFPE) tissue sections were incubated with Syn1 (1:1000; Fisher Scientific, Waltham, MA, USA) or EP1536Y (1:5000; Abcam, Cambridge, UK), developed with diaminobenzidine (DAB) chromagen, and counterstained with hematoxylin. Additionally, MSA cases were immunofluorescently labeled, and 7 μm FFPE sections from the pons and putamen were blocked in 5% BSA in PBS + 1% TritonX-100 (PBST) for 1 hr at room temperature before incubation overnight at 4 °C in EP1536Y (1:500; Abcam, Cambridge, UK) or Syn1 (1:500; Fisher Scientific, Waltham, MA, USA) diluted in 1% BSA PBST. Appropriate secondary antibodies (Alexa 555; 1:1000; Abcam, Cambridge, UK) and DAPI diluted in 1% BSA PBST were incubated for 1 hr at room temperature. Lipofuscin autofluorescence was quenched in 1× TrueBlack Plus (Biotium, Fremont, CA, USA). Immunohistochemical and immunofluorescent slides were imaged on a Zeiss ApoTome2 microscope and Zeiss LSM 980 confocal microscope, respectively.
4
Optogenetic Stimulation of DRG Neurons
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DRGs (L1–L6) were isolated from Vglut2-ChR2-YFP mice and cultured in poly-L lysine (100 µg/mL) and laminin (50 µg/mL) precoated chamber plates for 48 h. Cells were stimulated using 470 nm LED light (or control yellow light at 595 nm) at 20 Hz, and 10% duty cycle for 30 min using fiber-coupled LEDs (Thorlabs Inc). The supernatant was collected over time (for HMGB1 release) or for an additional 60-min incubation for measurements of LDH release (Sigma-Aldrich). For HMGB1 staining, cultured DRG cells were fixed with 4% paraformaldehyde (Thermo Fisher Scientific) followed by immunofluorescent staining with anti-HMGB1 antibody Alexa-555 (red, Abcam) and DAPI (blue) with endogenous VGlut2-YFP. Images were acquired using an LSM880 laser scanning confocal microscope (Zeiss). Levels of secreted HMGB1 in the supernatant were quantitated using an ELISA kit (IBL International).
5
Immunofluorescence Staining of Cellular Markers
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Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature. Samples were permeabilized, blocked, and incubated at 4 °C overnight with primary antibodies against GATA4 (1:200, Abcam, ab124265), PDGFR-α (1:250, Cell Signaling Technology, #3174), vimentin (1:500, Abcam, ab73159) or α-SMA–Cy3-conjugated (1:400, Sigma, C6198). Samples were then incubated for 1 h at room temperature with secondary antibodies conjugated with Alexa-488, Alexa-555, or Alexa-647 (1:500; Abcam). The cells were counterstained with Hoechst 33342 nuclear dye (1:500; Santa Cruz Biotechnology) and actiStain phalloidin conjugated to Alexa-670 (1:200; Cytoskeleton Inc.).
Images were captured under a laser confocal microscope (Nikon A1R, Nikon; RCM1, confocal.nl) and analysed on ImageJ (NIH).
Images were captured under a laser confocal microscope (Nikon A1R, Nikon; RCM1, confocal.nl) and analysed on ImageJ (NIH).
6
Quantification of Goblet and Ciliated Cells
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To detect the goblet and ciliated cells present in the cultures, antibodies were used to target specific cell type markers. Each culture was first fixed in 4% paraformaldehyde (Thermo Fisher Scientific) for 15 min and then blocked for 1 h in a blocking solution (0.5% Triton™ X-100 [Thermo Fisher Scientific], 5% normal goat serum [Thermo Fisher Scientific], and 2% bovine serum albumin [Thermo Fisher Scientific] in PBS). The cultures were stained with a β-tubulin 4 antibody (BTUB4; ciliated cell marker) conjugated to Alexa 647 (clone EPR16775, Abcam, Cambridge, United Kingdom) or a MUC5AC antibody (goblet cell marker) conjugated to Alexa 555 (clone EPR16904, Abcam), diluted in PBS with 2% normal goat serum and 1% bovine serum albumin. Finally, nuclei were counterstained with Hoechst 33342 dye (Thermo Fisher Scientific). For these MUC5AC- and BTUB4-stained slides, 4 and 16 contiguous fields of view located at the center of the inserts (see Fig. S1) were acquired, respectively. Images were acquired with the CellInsight™ CX7 high-content screening platform (Thermo Fisher Scientific), and positively stained cells were quantified as previously described (Marescotti et al., 2020 (link)).
7
Aortic Root Cryosectioning for Lipid and Collagen Analyses
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The aortic root was dissected under a microscope and frozen in optimal cutting temperature embedding medium for serial cryosectioning at 10 μm, covering 400 μm of the aortic root. The first section was collected when 3 aortic valve cusps became visible in the lumen of the aorta. Every tenth section was harvested on one slide (2 sections per slide). Lipids were detected by Oil Red O staining (Sigma). Collagen was stained with Sirius Red (Sigma). Section images of lipids were captured digitally by an Olympus BX53 imaging system (Olympus, Tokyo, Japan), and section images of collagen were captured digitally by Nikon Eclipse ci imaging system (Nikon, Tokyo, Japan). Macrophage content was analyzed by immunofluorescence staining with CD68 monoclonal antibody (CD68, 1 : 100, Abcam). Immunofluorescence staining was performed with primary antibodies to identify smooth muscle cells (α-SMA, 1 : 100, Abcam). Then, the sections were further incubated with secondary antibodies conjugated with Alexa 555 (Abcam). Nuclei were tagged with DAPI. The images were visualized and captured by using a microscope (Olympus, BX53).
8
Comprehensive Immunofluorescence Staining of Rat Kidneys
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Immunofluorescence staining was performed as described previously23 (link). Briefly, rat kidneys were fixed with 10% neutral buffered formalin, cryoprotected in 10% sucrose in PBS (6 h at 4 °C), immersed overnight in 20% sucrose in PBS at 4 °C, and frozen in OCT Tissue-Tek compound (SAKURA, Cat#: 4583) before 6 μm-thick cryosections were prepared. Anti-VEGFC (Immunoway, Cat#: YT5297), anti-CD68 (Abcam, Cat#: ab955), anti-LYVE-1 (Novus Biologicals, Cat#: NB600-1008), anti-α-SMA (Abcam, Cat#: ab202509), anti-vimentin (Abcam, Cat#: ab8978) and anti-collagen I (Abcam, Cat#: ab270993) were used to examined the frozen kidney sections. Secondary antibodies conjugated with Alexa 488 (Abcam, Cat#: ab150113, ab150081), Alexa 555 (Abcam, Cat#: ab150078, ab150106) and DyLight 405 (Abcam, Cat#: ab175651) were used to visualize antigen-antibody complexes. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Digital images were then obtained by confocal scanning microscopy (CTS SP8, Leica, Germany).
9
Immunostaining of Differentiated iMG Cells
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Monocytes were plated on PLL-coated coverslips in a 24-well flatbottom plate. On day ten of differentiation, iMG were washed, fixed with 4% PFA, and immunostained for IBA1 (rabbit, Wako, 019-19741), and CD68 (mouse, Invitrogen, MA5-13324). After incubation with the secondary antibodies (donkey anti-rabbit, Alexa555, Abcam, 150074); donkey anti-mouse (Delight488, ThermoFisher, A21202)) and nuclear staining (hoechst, ThermoFisher, H3569), immunostainings were imaged with Zeiss Axio-Scope A1.
10
Immunohistochemical Analysis of Nerve and Spinal Cord
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Animals were anesthetized and sacrificed on day 21 after implantation of cancer cells. The sciatic nerve eroded by tumor mass and spinal cord were removed and postfixed at 4 o C for 5 h with 4% paraformaldehyde/PBS (pH7.4). The postfixed sciatic nerve and spinal cord were transferred to 15% sucrose/PBS for 24 h and then 30% sucrose/PBS for 24 h. The samples were frozen at -80 o C, serially cut with a cryostat (15 μm) and mounted on silane-coated slides.
After washing with ice cold PBS, sciatic nerve and spinal cord were blocked in solution containing 10 % normal goat serum and 0.1% Triton X-100 for 2 h at 4 o C. The sections were then incubated at 4 o C with primary antibodies against CD11b (1:1000; Cat# MCA711G, AbD Serotec, Oxford, UK), Iba1 (1:1,000; Cat# 01919741, FUJIFILM Wako Pure Chemical Tokyo, Japan), CD68 (1:500, Cat# 137001, BioLegend, San Diego, CA), or myelin-basic protein (1:500, Cat# 836504, BioLegend) for 48 h. After washing, the sections were incubated with a fluorescent-conjugated secondary antibody (Alexa 488, Alexa 546, Alexa 555 or Alexa 647, 1:1,000; Abcam, Cambridge, UK) at 4 o C for 2 h. The slides were covered with one drop of Vectashield (Vector Laboratories, Burlingame, CA), and then cover-slipped. Fluorescent images were obtained with confocal fluorescence microscopy.
After washing with ice cold PBS, sciatic nerve and spinal cord were blocked in solution containing 10 % normal goat serum and 0.1% Triton X-100 for 2 h at 4 o C. The sections were then incubated at 4 o C with primary antibodies against CD11b (1:1000; Cat# MCA711G, AbD Serotec, Oxford, UK), Iba1 (1:1,000; Cat# 01919741, FUJIFILM Wako Pure Chemical Tokyo, Japan), CD68 (1:500, Cat# 137001, BioLegend, San Diego, CA), or myelin-basic protein (1:500, Cat# 836504, BioLegend) for 48 h. After washing, the sections were incubated with a fluorescent-conjugated secondary antibody (Alexa 488, Alexa 546, Alexa 555 or Alexa 647, 1:1,000; Abcam, Cambridge, UK) at 4 o C for 2 h. The slides were covered with one drop of Vectashield (Vector Laboratories, Burlingame, CA), and then cover-slipped. Fluorescent images were obtained with confocal fluorescence microscopy.
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