For the detection of BCMA-expressing cells in patients with PC neoplasia, PB slides were stained with the developed assay in patients with MGUS, SMM, NDMM, RRMM, and LPL. Slides were thawed after storage at −80 °C, fixed with 2% paraformaldehyde for 20 min, and washed using TBS. The slides were incubated with 10% goat serum for 30 min to block non-specific binding sites for secondary antibodies. Next, the slides were incubated with a primary antibody mix consisting of CD138 (2 µg/mL, Exbio), CD45-Alexa647 (1.6 µg/mL, AbD Serotec), and BCMA (2.5 µg/mL, Abcam) for 1 h at room temperature. The slides were then washed with TBS and subjected to an additional 1 h of incubation with 10% goat serum. The secondary antibodies Alexa Fluor 555 goat anti-mouse (1:500, Invitrogen) and Alexa FluorPLUS 488 goat anti-rabbit (1:500, Invitrogen) were added for the visualization of CD138 and BCMA, respectively, with DAPI for 40 min at room temperature. The slides were washed with TBS and rinsed in water prior to being mounted with live cell media, coverslipped, and sealed for downstream imaging and technical analysis.
Alexa fluor plus 488 goat anti rabbit
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor Plus 488 goat anti-rabbit is a fluorescently-labeled secondary antibody used for detection and visualization in various immunoassays and microscopy applications. It is designed to bind to and detect rabbit primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Ixplore spinsr scanr, by Olympus (2 mentions)
Fluoroshield with dapi, by Merck Group (2 mentions)
Alexa fluor 555 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
N storm a1 microscope system, by Nikon (1 mentions)
Goat anti mouse alexa fluor plus 594, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Alexa fluor 488 chicken anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 donkey anti sheep igg, by Thermo Fisher Scientific (1 mentions)
A21200, by Thermo Fisher Scientific (1 mentions)
Nunc lab tek 2 chamber slide system, by Thermo Fisher Scientific (1 mentions)
Nunc lab tektm 2 chamber slide system, by Thermo Fisher Scientific (1 mentions)
5 protocols using alexa fluor plus 488 goat anti rabbit
1
BCMA-Expressing Cell Detection in PC Neoplasia
2
Immunofluorescence Imaging of XPa-Stimulated Cells
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For immunofluorescence assays, cells were plated into 6-well plates and stimulated with XPa at an MOI of 10 for 24 h. Then the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS). After permeabilization with 0.1% Triton X-100 (Sigma) in PBS, the cells were blocked in a solution containing 2.5% BSA (Sigma) and 2.5% goat serum for 1 h and then stained with specific primary antibodies (CST) (1:200) followed by secondary antibodies (1:200) (Alexa Fluor Plus 488 goat anti-rabbit and Alexa Fluor Plus 594 goat anti-mouse; Invitrogen, Thermo Fisher Scientific). Finally, the cells were stained with DAPI, and the slides were sealed with antifluorescence-quenching sealing tablets. Stochastic optical reconstruction microscopy (STORM) and total internal reflectance fluorescence (TIRF) images were obtained on the N-STORM&A1 microscope system (Nikon).
3
Fluorescent Antibody Labeling Protocol
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Alexa Fluor 488 chicken anti-mouse immunoglobulin G (IgG) (Invitrogen #A21200, RRID_AB_2535786, used at 1:400); Alexa Fluor 488 chicken anti-rabbit IgG (Invitrogen #A21441, RRID_AB_10563745, used at 1:400); Alexa Fluor 568 donkey anti-sheep IgG (Invitrogen #A21099, RRID_AB_10055702, used at 1:400); Alexa Fluor Plus 488 goat anti-rabbit (ThermoFisher A32731, used at 1:500); Alexa Fluor Plus 594 goat anti-mouse (ThermoFisher A32742, used at 1:500); and goat anti-mouse IgG-HRP (horseradish peroxidase) (BioRad #170-6516, RRID:AB_11125547, used at 1:20000).
4
Evaluating Autophagy via LC3A/B Staining
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Cells were seeded on 8-well chamber slide (Nunc™ Lab-Tek™ II Chamber Slide™ System/ Thermo Fisher Scientific Inc, Waltham, MA, USA) and reached to get 80% confluence. Then, cells were treated with DON and LY294002 for 24 h. After incubation time, the experimental medium was discarded and fixed with 70% iced methanol for 15 min in freezer. Next, cells were washed three times in dPBS, blocked (5% FBS, 0.3% Triton X-100 in dPBS 1X) for 1 h and then incubated in primary antibodies against LC3A/B (1:100, 1% BSA, 0.3% Triton X-100 in dPBS 1X. #12,741, Cell Signaling Technology) overnight. The next day, cells were washed in dPBS three times and incubated 1.5 h with secondary antibodies (1:400, Alexa Fluor Plus® 488, goat anti-rabbit #A32731, Thermo Fisher Scientific Inc, Waltham, MA, USA). Then, cells were washed three times in dPBS, walls were removed and slide was sticked down using Fluoroshield™ with DAPI (F6057, Sigma, St. Louis, MO, USA). Olympus iXplore SpinSR ScanR was used for visualization (Olympus, Tokyo, Japan).
5
Immunofluorescence Analysis of Caveolin-1
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The cells were seeded on 8-well chamber slides (NuncTM Lab-TekTM II Chamber SlideTM System/Thermo Fisher Scientific Inc, Waltham, MA, USA). Once the slides reached 80% confluence, experimental treatments were performed for 48 h. Cell fixation was performed in 70% ice-cold methanol for 15 min in a freezer. The cells were washed three times in DPBS. Subsequently, cells were immersed in the blocking buffer (5% FBS, 0.3% Triton X-100 in DPBS 1X) for 1 h. Incubation with the primary antibody CAV-1 (sc-894, Santa Cruz Biotechnology, Dallas, TX, USA) was performed overnight (1:100, 1% BSA, 0.3% Triton X-100 in DPBS 1X). The following day, cells were washed three times with DPBS and incubated for 1.5 h with a secondary antibody (1:400, Alexa Fluor Plus® 488, goat anti-rabbit, #A32731, Thermo Fisher Scientific Inc, Waltham, MA, USA). Upon completed incubation, the final washing step was performed (three times in DPBS 1X), and the mounting medium, Fluoroshield with DAPI (F6057, Sigma, St. Louis, MO, USA), was used to preserve the fluorescence of cell specimens. Confocal fluorescence imaging was performed with the use of Olympus iXplore SpinSR ScanR (Olympus, Tokyo, Japan).
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