We performed an immunofluorescence assay to detect the presence and titer of antibodies against SARS-CoV-2 spike and nucleocapsid proteins, as described previously [36] (link). The transfected HEK-293T cells (ATCC, USA) that expressed either the spike (Addgene, USA) or nucleocapsid (Sini Biological, USA) proteins of SARS-CoV-2 were fixed and seeded onto 12-well polytetrafluoroethylene printed slides (Electron Microscopy Sciences, USA). After a 1: 20 dilution in 1X PBS with 0.1% Tween-20, the plasma was added to the corresponding wells on the slide and incubated for 1 hour at 37°C. The slides were then washed and incubated with secondary mouse monoclonal antihuman immunoglobulin G antibody (ATCC, USA), with subsequent removal of unbound antibodies and incubation with tertiary CY2-conjugated donkey antimouse immunoglobulin G (Jackson Immuno Research Laboratories, USA). This was then followed by counterstaining the cells with Evans blue dye. For the titration, the serial dilutions on positive samples were done beginning from 1: 20 until a negative reading. The reading of the slides was done by three independent readers on a Nikon E400 fluorescence microscope to determine positive or negative signals, and only harmonious results from at least two independent readers were reported as the outcome.
E400 fluorescence microscope
Manufactured by Nikon
Sourced in Japan
The Nikon E400 fluorescence microscope is a laboratory instrument designed for the analysis of fluorescently labeled samples. It features powerful illumination and high-quality optics to provide clear and detailed images of fluorescent specimens. The core function of the E400 is to enable researchers and scientists to visualize and study fluorescent-labeled samples with precision and accuracy.
Automatically generated - may contain errors
Lab products found in correlation
Hek293t cell, by American Type Culture Collection (1 mentions)
Alexa fluor 594 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Trs antigen retrieval solution, by Agilent Technologies (1 mentions)
Autostainer, by Agilent Technologies (1 mentions)
Labview, by National Instruments (1 mentions)
Alexa fluor 488 conjugated anti goat igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated anti goat igg, by Thermo Fisher Scientific (1 mentions)
Nis elements imaging software, by Nikon (1 mentions)
Gtx110624, by GeneTex (1 mentions)
5 protocols using e400 fluorescence microscope
1
SARS-CoV-2 Antibody Detection Assay
2
Immunofluorescence Staining of FANC-D2 and Ki67
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The FA triple staining immunofluorescence method has been previously described [22 (link)]. Briefly, FFPE tumor tissue was cut at 4 microns, placed on positively charged slides, and stained with hematoxylin and eosin. Additional sections for immunofluorescence staining were placed in a 60 °C oven for one hour, cooled, deparaffinized, and rehydrated through xylenes and graded ethanol solutions to water in standard fashion. Antigen retrieval was performed by placing slides in Dako’s TRS antigen retrieval solution (Dako) in a calibrated vegetable steamer (Black and Decker) at 94 °C for 25 min. Slides then were placed on a Dako Autostainer for automated staining. The tissue sections were incubated with a primary antibody cocktail of rabbit polyclonal FANC-D2 antibody at a dilution of 1:1,000 and a monoclonal anti-Ki67 mouse antibody (Dako) at a dilution of 1:150, for one hour at room temperature. Sections then were incubated with a secondary antibody (FITC conjugated to anti-rabbit IgG and Alexa fluor 594 donkey anti-mouse IgG, Invitrogen) at 1:1,000 for one hour at room temperature. The sections were mounted on glass slides using a 4′ 6-diamidino-2-phenylindole (DAPI)-containing embedding medium (Vysis Dapi 1, Abbott Laboratories). The slides were analyzed under a Nikon E-400 fluorescence microscope [22 (link)].
3
Multi-color Fluorescence Microscopy Imaging
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A Nikon E400 fluorescence microscope equipped with a Mercury Arc lamp as light source, a 10X (0.45NA) objective (Nikon, Tokyo, Japan), a computer-controlled CCD camera (Hamamatsu Photonics, Hamamatsu, Japan), X,Y,Z stage and 4-filter cube exchanger (LEP, Hawthorne NY, USA) with filters were used for acquisition of the fluorescent images covering the 0.64 cm2 surface of the microsieves. The following filters were used: APC with excitation 621/32 nm, dichroic 647 nm LP, emission 682/52 nm (Spectra Physics Newport, Santa Clara, CA, USA); BV421 with excitation 390/40 nm, dichroic 405 nm LP, emission 438/24 nm (Semrock, Rochester, NY, USA); FITC with excitation 480/20 nm, dichroic 495 nm LP, emission 510/20 nm (Spectra Physics Newport); and PE with excitation 547/12 nm, dichroic 560 nm LP, emission 579/25 nm (Spectra Physics Newport). The scanning and image acquisition was controlled by Labview (National Instruments, Austin TX, USA).
4
Immunofluorescence Staining of Frozen Sections
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Frozen sections were probed with primary antibody in PBS/ 0.01% Triton X-100 at 4 °C overnight. All primary antibodies were listed in Supplementary Table 2. The sections were then incubated with Alexa Fluor 488-conjugated anti-goat IgG or Alexa Fluor 594-conjugated anti-goat IgG (Invitrogen) secondary antibodies for 2 h. Images were acquired on a Nikon E400 fluorescence microscope (Tokyo, Japan). Digitally captured images were analyzed using NIS-Elements imaging software (Nikon, Tokyo, Japan).
5
Quantifying Oxidative Stress in Kidney Tissues
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Paraffin-embedded kidney sections (4 μm) were processed for immunostaining by deparaffinization in xylene and rehydration through a descending series of alcohols, washed extensively in 0.1 M PBS, blocked with 10% normal goat serum, and incubated in primary antibodies to 8-hydroxy-2'deoxyguanosine (8-OHdG) (Abcam Cat# ab48508, RRID:AB_867461 ) or citrate synthase (GeneTex Cat# GTX110624, RRID:AB_1950045 ) diluted in 10% normal goat serum overnight at 4°C, followed by fluorescent-labeled secondary antibodies labeled with Alexa Fluor 488 green or Alexa Fluor 556 red. Nuclei were counterstained with DAPI. Images were acquired on a Nikon E400 fluorescence microscope.
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