Indirect immunofluorescence was performed as described previously.96 (link) First, cells were harvested by centrifugation and rinsed with PBS buffer twice. Next, 100 μL of cells were spotted onto Poly-L-lysine-coated glass slides (Sigma-Aldrich). Cell fixation was done by 4 % (w/v) formaldehyde (Sigma-Aldrich) in PBS for 20 min and then incubated with 100 % ice-cold methanol for 20 min to remove chlorophyll. Purified antibodies (Yenzyme) against PMR1 were used at a dilution of 1:50. The purified antibodies were generated using the following peptide: C-Ahx-EGRSFQDDSTGREQSQGY-amide. After washing the slides six times, each with 50 mL PBS-T (with 0.1% Tween 20 (v/v)) in a Coplin jar, Alexa Fluor 488 goat anti-rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (Invitrogen) was used at a dilution of 1:500. The slides were washed six times, each with 50 mL PBS-T. Fluorescence and bright-field images were acquired using a confocal microscope (Leica, SP5).
Alexa fluor 488 goat anti rabbit igg h l cross adsorbed secondary antibody
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488 goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody is a fluorescently labeled secondary antibody that binds to rabbit primary antibodies. It is used to detect and visualize rabbit immunoglobulins (IgG) in various applications such as immunohistochemistry, flow cytometry, and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Sapphire biomolecular imager, by Azure Biosystems (5 mentions)
Poly l lysine coated glass slide, by Merck Group (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Cross reactive rabbit anti sars cov 2 n igg, by Sino Biological (1 mentions)
7 protocols using alexa fluor 488 goat anti rabbit igg h l cross adsorbed secondary antibody
1
Indirect Immunofluorescence of PMR1 Protein
2
Immunofluorescence Analysis of Neural Cell Markers
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LOCS seeded with in vitro cultured cells were fixed with 4 % paraformaldehyde (PFA) and sectioned after paraffin embedding. Spinal cord samples from the rats were removed immediately after infusion with 4 % PFA and sectioned after paraffin embedding. The following antibodies were used for immunofluorescence: Nestin (Proteintech, 29285-1-AP, 1:200), Sox2 (Proteintech, 11064-1-AP, 1:200), TrkC (Proteintech, 66380-1-Ig, 1:100), HNA (Abcam, ab191181, 1:100), GFAP (Proteintech, 16825-1-AP, 1:200), Tuj-1 (Abcam, ab18207, 1:100), Map-2 (Proteintech, 17490-1-AP, 1:100), NeuN (Abcam, ab177487, 1:100), MBP (Proteintech, 10458-1-AP, 1:100), Syn (Proteintech, 17785-1-AP, 1:100), PSD95 (Proteintech, 20665-1-AP, 1:100), 5-HT (Sigma-Aldrich, S5545, 1:1000), ChAT (Proteintech, 20747-1-AP, 1:200), VGLUT1 (Proteintech, 55491-1-AP, 1:200), CD68 (Abcam, ab283654, 1:100), NG2 (Abcam, ab275024, 1:100), Laminin (Proteintech, 23498-1-AP, 1:200), Integrin beta 1 (Abcam, ab179471, 1:100). Alexa Fluor™ 488 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (Invitrogen, A-11008, 1:500); Alexa Fluor™ 594 Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody (Invitrogen, A-11005, 1:500). DAPI (Beyotime, C1002, 1:500) was used for nuclear staining.
3
SARS-CoV-2 Neutralization Assay via FRNT
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The SARS-CoV-2 focus reduction neutralization test (FRNT) was performed in a certified biosafety level 3 laboratory. Neutralization assays against live SARS-CoV-2 were conducted using a clinical isolate (HKU-001a strain, GenBank accession no: MT230904.1) previously obtained from a nasopharyngeal swab of an infected patient (Chu et al., 2020 (link)). Serial dilutions of testing antibodies were conducted, mixed with 50 μL of SARS-CoV-2 (1 × 103 focus forming units/mL, FFU/mL) in 96-well plates and incubated for 1 h at 37°C. Mixtures were then transferred to 96-well plates preseeded with 1 × 104/well Vero E6 cells and incubated at 37°C. After 24 h, the culture medium of the plates was removed and air-dried in a biosafety cabinet (BSC) for 20 min. Cells were then fixed with 4% paraformaldehyde solution for 30 min and air-dried in BSCs again. Cells were further permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit anti-SARS-CoV-2-N IgG (Sino Biological, Inc.) for 1 h at RT before adding Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Life Technologies). The numbers of SARS-CoV-2 foci were calculated using the Sapphire Biomolecular Imager (Azure Biosystems).
4
SARS-CoV-2 Neutralization Assay in HEK293T-CD209 Cells
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HEK293 T cells were seeded into 10-cm dish at 40% confluency and cultured overnight. The HEK293 T cells were transfected with human CD209 (Sino Biological) at 70–90% confluency. The expression of CD209 was measured by flow cytometry. The transfected HEK293T-CD209 cells were seeded into 96-well plates with 2.4 × 104 cells per well and cultured overnight. The HEK293T-CD209 cells were pre-treated with 10 ng/ml of B8-dIgA or control dIgA and incubated for 6 h prior SARS-CoV-2 infection (MOI = 0.05). 24 h later, cells were then fixed with 4% paraformaldehyde solution for 30 min and air-dried in the BSC. Cells were further permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit sera anti-SARS-CoV-2-N for 1 hour at RT before adding Alexa Fluor 488 goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody (Life Technologies). The fluorescence density of SARS-CoV-2 infected cells was acquired using a Sapphire Biomolecular Imager (Azure Biosystems) and then the MFI of four randomly selected areas of each sample was quantified using Fiji software (NIH).
5
SARS-CoV-2 Microneutralization Assay Protocol
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Live virus microneutralization assay was performed as we described previously 58 (link). Briefly, serum specimens were serially diluted in 3-folds from 1:10. Equal volumes of serially diluted sera and the desired SARS-CoV-2 strains (1 × 103 PFU/mL) were mixed, added onto 96-well plates and incubated for 1 h at 37oC. Mixtures were then transferred to 96-well plates pre-seeded with 2 × 104/well Vero E6 cells and incubated at 37oC overnight. The culture medium of the plates was removed, air-dried in a biosafety cabinet (BSC) for 20 min. Cells were then fixed with 4% paraformaldehyde solution. To further permeabilize the cells, 100 μL of 0.2% Triton X-100 in PBS was added to each well and the plates were incubated at RT for 15 min. After three washes in PBS, the plates were incubated with cross-reactive rabbit anti-SARS-CoV-2 N IgG (diluted 1:4000 in 0.2% Triton X-100 in PBS) for 1 h, followed by the addition of Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (diluted 1:1000 in 0.2% Triton X-100 in PBS, Life Technologies) and incubation at RT for 1 h. The plates were analyzed using Sapphire Biomolecular Imager (Azure Biosystems). The numbers of SARS-CoV-2 foci were quantified using custom-built ReadPlate 3.0 plugin in ImageJ 59 (link).
6
SARS-CoV-2 Focus Reduction Neutralization Assay
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The SARS-CoV-2 focus reduction neutralization test (FRNT) was performed in a certified Biosafety level 3 laboratory. Neutralization assays against live SARS-CoV-2 were conducted using a clinical isolate previously obtained from a nasopharyngeal swab from an infected patient60 (link). The tested antibodies were serially diluted, mixed with 50 μL of SARS-CoV-2 (1 × 103 focus forming unit/mL, FFU/mL) in 96-well plates, and incubated for 1 hour at 37 °C. Mixtures were then transferred to 96-well plates pre-seeded with 1 × 104/well Vero-E6 cells and incubated at 37 °C for 24 h. The culture medium was then removed, and the plates were air-dried in a biosafety cabinet (BSC) for 20 min. Cells were then fixed with a 4% paraformaldehyde solution for 30 min and air-dried in the BSC again. Cells were further permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit sera anti-SARS-CoV-2-N (1:5000) for 1 h at RT before adding an Alexa Fluor 488 goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody (1:1000 Life Technologies). The fluorescence density of SARS-CoV-2 infected cells were scanned using a Sapphire Biomolecular Imager (Azure Biosystems) and the neutralization effects were then quantified using Fiji software (NIH).
7
Quantitative SARS-CoV-2 Neutralization Assay
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The SARS-CoV-2 focus reduction neutralization test (FRNT) was performed in a certified Biosafety level 3 laboratory as previously described [21 (link)]. The tested antibodies were serially diluted, mixed with 50 μL of SARS-CoV-2 (103 focus forming unit/mL, PFU/mL) in 96-well plates, and incubated for 1 h at 37°C. Mixtures were then transferred to 96-well plates pre-seeded with 2 × 104/well Vero-E6-TMPRSS2 cells and incubated at 37°C for 24 h. The culture medium was then removed and then fixed with a 4% paraformaldehyde solution for 30 min and air-dried in the BSC again. Cells were further permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit sera anti-SARS-CoV-2-N (1:5000) for 1 h at RT before adding an Alexa Fluor 488 goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody (1:1000 Life Technologies). The fluorescence density of SARS-CoV-2 infected cells were scanned using a Sapphire Biomolecular Imager (Azure Biosystems) and the neutralization effects were then quantified using Fiji software (NIH).
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