Peripheral blood mononuclear cells (PBMCs) and the single-cell sorting strategy were performed as previously described5 (link). In brief, PBMCs were isolated from heparin-treated whole blood by density gradient centrifugation (Ficoll-Paque PREMIUM, Sigma-Aldrich). After separation, PBMCs were stained with Live/Dead Fixable Aqua (Invitrogen; Thermo Scientific) diluted 1:500 at room temperature. After 20 min of incubation, cells were washed with PBS and unspecific bindings were saturated with 20% normal rabbit serum (Life Technologies). Following 20 min of incubation at 4 °C, cells were washed with PBS and stained with SARS-CoV-2 S protein labelled with Strep-Tactin XT DY-488 (2-1562-050, iba-lifesciences) for 30 min at 4 °C. After incubation, the following staining mix was used CD19 V421 (1:320; 562440, BD), IgM PerCP-Cy5.5 (1:50; 561285, BD), CD27 PE (1:30; 340425, BD), IgD-A700 (1:15; 561302, BD), CD3 PE-Cy7 (1:100; 300420, BioLegend), CD14 PE-Cy7 (1:320; 301814, BioLegend), CD56 PE-Cy7 (1:80; 318318, BioLegend) and cells were incubated at 4 °C for an additional 30 min. Stained memory B cells were single-cell-sorted with a BD FACS Aria III (BD Biosciences) into 384-well plates containing 3T3-CD40L feeder cells and were incubated with IL-2 and IL-21 for 14 days as previously described25 (link).
Normal rabbit serum
Manufactured by Thermo Fisher Scientific
Sourced in United States
Normal rabbit serum is a laboratory reagent used as a control or blocking agent in various immunological techniques, such as immunohistochemistry, Western blotting, and ELISA. It is derived from the blood of healthy rabbits and contains a diverse array of natural antibodies and proteins found in rabbit serum.
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Lab products found in correlation
Superfrost plus slide, by Thermo Fisher Scientific (8 mentions)
Camedia c 2000 digital camera, by Olympus (3 mentions)
Bx40 microscope, by Olympus (3 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions)
Live dead fixable aqua, by Thermo Fisher Scientific (2 mentions)
Ficoll paque premium, by Merck Group (2 mentions)
Strep tactin xt dy 488, by IBA Lifesciences (2 mentions)
Facsaria 3, by BD (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Anti hmgb1 rabbit polyclonal antibodies, by Novus Biologicals (2 mentions)
Anti asialo gm1, by Fujifilm (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Igm percp cy5, by BD (1 mentions)
Cd56 pe cy7, by BioLegend (1 mentions)
Igd a700, by BD (1 mentions)
Cd14 pe cy7, by BioLegend (1 mentions)
Cd3 pe cy7, by BioLegend (1 mentions)
Cd27 pe, by BD (1 mentions)
Aec substrate kit, by Merck Group (1 mentions)
Cm1860 uv, by Leica Microsystems (1 mentions)
21 protocols using normal rabbit serum
1
Isolation and Single-Cell Sorting of SARS-CoV-2 Reactive Memory B Cells
2
Isolation and Expansion of SARS-CoV-2-Specific B Cells
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Blood samples were screened for SARS-CoV-2 RNA and for antibodies against HIV, HBV and HCV. Peripheral blood mononuclear cells (PBMCs) were isolated from heparin-treated whole blood by density gradient centrifugation (Ficoll-Paque PREMIUM, Sigma-Aldrich). After separation, PBMC were stained with Live/Dead Fixable Aqua (Invitrogen; Thermo Scientific) in 100 μL final volume diluted 1:500 at room temperature RT. After 20 min incubation cells were washed with PBS and unspecific bindings were saturated with 50 μL of 20% normal rabbit serum (Life technologies) in PBS . Following 20 min incubation at 4°C cells were washed with PBS and stained with SARS-CoV-2 S-protein labeled with Strep-Tactin®XT DY-488 (iba-lifesciences cat# 2-1562-050) for 30 min at 4°C. After incubation the following staining mix was used CD19 V421 (BD cat# 562440), IgM PerCP-Cy5.5 (BD cat# 561285), CD27 PE (BD cat# 340425), IgD-A700 (BD cat# 561302), CD3 PE-Cy7 (BioLegend cat# 300420), CD14 PE-Cy7 (BioLegend cat# 301814), CD56 PE-Cy7 (BioLegend cat# 318318) and cells were incubated at 4°C for additional 30 min. Stained MBCs were single cell-sorted with a BD FACS Aria III (BD Biosciences) into 384-well plates containing 3T3-CD40L feeder cells and were incubated with IL-2 and IL-21 for 14 days as previously described (Huang et al., 2013 (link)).
3
Immunohistochemical Analysis of Toll-like Receptor 4
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The transverse colon was embedded in Tissue-Tek (Sakura, Tokyo, Japan), snap-frozen in isopentane, cooled in liquid nitrogen vapor, and stored at −70 °C. Then, 5-μm acetone-fixed cryosections were cut on a cryostat CM 1860 UV (Leica Microsystems, Wetzlar, Germany) and put on SuperFrost/Plus slides (Thermo Fisher Scientific, Darmstadt, Germany) and were kept at −40 °C until labeling. The sections were incubated with 10% normal rabbit serum (Life Technologies, Carlsbad, CA, USA) in a humid chamber for one h at RT. Labeling by anti-TLR4 rabbit polyclonal antibodies (Novus Biologicals, Centennial, CO, USA) was performed overnight at 4 °C. The sections were incubated with secondary antibody, peroxidase-conjugated F(ab’)2-goat anti-rabbit IgG (H+L) (Life Technologies, Carlsbad, CA, USA) for 2 h at RT. The TLR4 localization was visualized by an incubation with AEC substrate kit (Sigma-Aldrich, St. Louis, MO, USA) and examined under an Olympus BX 40 microscope with Olympus Camedia C-2000 digital camera (Olympus, Tokyo, Japan). Control sections without primary antibody were treated in the same way.
4
Immunofluorescence Analysis of Tight Junctions
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Specimens for immunofluorescence observation were embedded in Tissue-Tek (Sakura, Tokyo, Japan), snap frozen in isopentane cooled in liquid nitrogen vapor, and stored at −70°C for further processing. 5-µm acetone-fixed cryosections on SuperFrost/Plus slides (Thermo Fisher Scientific, Darmstadt, Germany) were kept at −40°C until labeling. Sections were incubated with normal rabbit serum (Life Technologies, Carlsbad, CA, USA) for 1 h at RT. Labeling by anti-human claudin-1 and anti-human occludin rabbit polyclonal antibodies (both Life Technologies) was performed overnight at +4°C. The sections were incubated with secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies), for 2 h at RT. The sections were subsequently embedded in ProLong Gold Antifade Reagent (Life Technologies). Control sections were treated in the same way, with the omission of primary antibody.
5
Immunohistochemical Analysis of HMGB1
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Amniotic membranes were embedded in Tissue-Tek (Sakura, Tokyo, Japan), snap-frozen in isopentane cooled in liquid nitrogen vapor and stored at −70 °C. Five μm acetone-fixed cryosections were cut on a cryostat CM1860 UV (Leica Microsystems, Wetzlar, Germany), put on SuperFrost/Plus slides (Thermo Fisher Scientific, Darmstadt, Germany), and were kept at −40 °C until labeling. Later, the sections were processed as described elsewhere [33 (link)]. Briefly, they were blocked with 10% normal rabbit serum (Life Technologies, Carlsbad, CA, USA) for 1 h at RT, incubated with anti-HMGB1 rabbit polyclonal antibodies (Novus Biologicals, Centennial, CO, USA) for 16 h at 4 °C, and labeled with Alexa Fluor 488-conjugated goat anti-rabbit IgG antibodies (Life Technologies) for 2 h at RT After embedding in ProLong Gold Antifade Reagent (Life Technologies), the sections were evaluated under an Olympus BX 40 microscope with an Olympus Camedia C-2000 digital camera (Olympus, Tokyo, Japan). The sections without primary antibodies served as controls. The HMGB1 and nuclei colocalization was verified by ImageJ software [34 (link)].
6
Immunofluorescence Localization of HMGB1
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Mesenteric lymph nodes were embedded in Tissue-Tek (Sakura, Tokyo, Japan), snap-frozen in isopentane cooled in liquid nitrogen vapor, and stored at −70 °C. 5-μm acetone-fixed cryosections on SuperFrost/Plus slides (Thermo Fisher Scientific, Darmstadt, Germany) were kept at −40 °C until labeling. The sections were incubated with 10% normal rabbit serum (Life Technologies, Carlsbad, CA, USA) in a humid chamber for one h at RT. Labeling by anti-HMGB1 rabbit polyclonal antibodies (Novus Biologicals, Centennial, CO, USA) was performed overnight at 4 °C. The sections were incubated with secondary antibody, Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies), for 2 h at RT. The sections were subsequently embedded in ProLong Gold Antifade Reagent (Life Technologies) and examined under an Olympus BX 40 microscope with a Olympus Camedia C-2000 digital camera (Olympus, Tokyo, Japan). Control sections without primary antibody were treated in the same way. The colocalization of HMGB1 and nuclei was analyzed by ImageJ software [114 (link)].
7
Olfactory Marker Protein Detection Protocol
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Anti-OMP was used as a molecular marker of mature olfactory chemosensory neurons. Olfactory marker protein was detected using polyclonal goat anti-OMP (dilution 1:10,000; Wako, Richmond, VA, USA; #019-2229 RRID: AB_664696). After incubation for 20 min with 1% normal rabbit serum (Life Technologies), sections were rinsed in anti-OMP at 4°C, overnight, in a humid chamber. After many washes in PBS the sections were incubated in rabbit anti-goat biotin-conjugated secondary antibody (dilution 1:200; Thermo Fisher Scientific) for 1 h at room temperature.
8
NK Cell Depletion and Characterization in Mice
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Two different NK-depleting antibodies, anti-asialo GM1 (Wako) and anti-CD122 (BioLegend), were tested for their ability to deplete pNKs and/or uNKs in vivo in order to generate mice deficient in both MCs and NKs. The efficiency of pNK depletion was analyzed by flow cytometry. The presence or absence of uNKs was assessed by uNK-specific Dolichos biflorus agglutinin (DBA) lectin staining of implantations at gd10. For the anti-asialo GM1 treatment, C57BL/6J females were mated and anti-asialo GM1 (100 μL, 0.29 mg) was administered i.p. at gd0, 2, 4, 6 and 8. Control females received 100 μL of normal rabbit serum (Life Technologies) at the same time points. For the anti-CD122 treatment, anti-CD122 (250 μL, 0.25 mg) was administered i.p. to C57BL/6J females once at gd0, immediately after the detection of the copulation plug. Control females received PBS (250 μL), as previously described59 (link)60 (link). Mice were sacrificed at gd10 or gd18 and different organs were analyzed by flow cytometry to assess the pNKs; implantation sections from gd10 were analyzed by DBA lectin-specific staining to assess uNKs.
9
NK Cell Depletion Protocol for Tumor Studies
10
Leptospira spp. Isolation and Identification
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Fifteen references Leptospira spp. including pathogenic, intermediate and non-pathogenic groups (Table 1 ); 101 of Leptospira spp. isolates and one isolate of Turneriella parva, a spirochete closely related to Leptospira, were included in this study (Table 2 ). Leptospira were cultured in Ellinghausen-McCullough-Johnson-Harris liquid medium containing Leptospira Medium Base EMJH (Difco, Becton Dickinson, USA), Leptospira Enrichment EMJH (Difco) with 3% normal rabbit serum (Invitrogen, USA) at 30°C. All Leptospira from human clinical samples were isolated and cultured as described [11 (link)]. Serovar identification was performed using cross-agglutination absorption test (CAAT) at WHO/FAO/OIE Collaborating Center for Reference and Research on Leptospirosis, Brisbane, Queensland, Australia. All clinical samples were identified at specie level based on sequencing of 16S rRNA gene as described [12 (link), 13 (link)].
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