Organs were collected from WT, CD45.1, CD45.2, OT-I, or CD8β-deficient mice and single-cell suspensions prepared using standard protocols. Following removal of red blood cells using ACK, nonspecific receptors were blocked with monoclonal antibody (mAb) 2.4G2, before cells (1–5 × 106) were stained with mAb to mouse NKp46 (29A1.4; BioLegend), CD3 (17A2; BD Biosciences), TCRγδ (GL3, BioLegend), TCRαβ (H57-597, BioLegend), CD8α (53-6.7; BD Biosciences), CD8β (H35-17.2; BD Biosciences). Alternatively cells were stained with the lectins peanut agglutinin (Vector Laboratories), sambucus nigra lectin (Vector Laboratories), or maackia amurensis lectin II (Vector Laboratories) before detecting using Streptavidin (BD Biosciences). Cells stained with tetramers were fixed in 2% paraformaldehyde for 15 min and washed twice with FACS buffer (1% FCS/PBS) before being resuspended in FACS buffer. All other FACS combinations were acquired unfixed. For acquisition, events were electronically gated on FSC-A versus FSC-H (singlets), followed by FSC-A and SSC-A (to exclude doublets and debris). Among the remaining population, at least 5000 electronic events of interest were collected using an LSR-II or X- 20 Fortessa (BD Biosciences).
Peanut agglutinin
Manufactured by Vector Laboratories
Sourced in United States
Peanut agglutinin is a lectin derived from the peanut plant (Arachis hypogaea). It is a carbohydrate-binding protein that specifically recognizes and binds to glycoproteins containing terminal galactose or N-acetylgalactosamine residues. Peanut agglutinin is commonly used in research applications as a tool for the detection and isolation of these types of glycoproteins.
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Lab products found in correlation
Superfrost plus slide, by Thermo Fisher Scientific (5 mentions)
B220 ra3 6b2, by Thermo Fisher Scientific (4 mentions)
Wheat germ agglutinin (wga), by Vector Laboratories (3 mentions)
Tissue tek oct, by Sakura Finetek (3 mentions)
Sambucus nigra lectin, by Vector Laboratories (2 mentions)
Galanthus nivalis lectin, by Vector Laboratories (2 mentions)
Tissue tek oct compound, by Sakura Finetek (2 mentions)
Axio imager m1 microscope, by Zeiss (2 mentions)
Cd5 53 7.3, by Thermo Fisher Scientific (2 mentions)
Metallophilic macrophages 1 moma 1, by Abcam (2 mentions)
Cd35 8c12, by BD (2 mentions)
Igm 2 41, by Thermo Fisher Scientific (2 mentions)
Ec plan neofluar objective, by Zeiss (2 mentions)
Cy3 streptavidin, by Jackson ImmunoResearch (2 mentions)
Cd3e 145 2c11, by BD (2 mentions)
Cd8α 53 6.7, by BD (1 mentions)
Cd3 17a2, by BD (1 mentions)
Tcrγδ gl3, by BioLegend (1 mentions)
Maackia amurensis lectin 2, by Vector Laboratories (1 mentions)
Fortessa x20, by BD (1 mentions)
23 protocols using peanut agglutinin
1
Multiparameter Flow Cytometry Analysis of Immune Cell Populations
2
Fluorescent Lectin Labeling Protocol
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The six fluorescein-labeled lectins Galanthus nivalis lectin (GNL; n = 8), Lens culinaris agglutinin (LCA; n = 5), Peanut agglutinin (PNA; n = 8), Solanum tuberosum lectin (STL; n = 8), Ulex europaeus agglutinin 1 (UEA I; n = 8), and Wheat germ agglutinin (WGA; n = 8) were purchased from Vector Laboratories (Burlingame, CA, USA). The lectin working solutions were prepared by diluting the stock solutions with simulated nasal electrolyte solution (SNES) to a final lectin concentration of 500 nmol/L. The lectin concentration was chosen based on a study by Engleder, Demmerer, Wang, Honeder, Zhu, Studenik, Wirth, Arnoldner, and Gabor [16 (link)] in a guinea pig model. The SNES contained 7.45 mg/mL of sodium chloride, 1.29 mg/mL of potassium chloride, and 0.32 mg/mL of calcium chloride dihydrate in distilled water [30 (link)]. All chemicals used for the preparation of SNES were of analytical grade and purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). This solution also served as a washing solution in the experiment. A solution of fluorescein isothiocyanate-conjugated bovine serum albumin (F-BSA; F/P ratio = 7–12; Sigma-Aldrich Corporation, St. Louis, MO, USA) in SNES (500 nmol/L) was used to rule out any non-specific protein binding. For lectin properties (source, molecular weight, binding motif, and molar fluorescein/protein (F/P) ratio), see Table 1 .
3
Immunofluorescent Staining and H&E Analysis
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Tissues were frozen in Tissue-TEK OCT (Sakura Finetek) compound at −80°C. For immunofluorescent staining, 6-µM sections were mounted on Superfrost Plus slides (Thermo Fisher Scientific), fixed with cold acetone for 10 min, and blocked with 5% FBS. The following antibodies were used to stain the sections: peanut agglutinin (Vector Labs) and B220 (RA3-6B2; Thermo Fisher Scientific) for 2 h at room temperature and washed with a 0.5% Tween in PBS (vol/vol) solution. Images were acquired using a Zeiss Axio ImagerM1 microscope and Slidebook software (Intelligent Imaging Innovations).
For H&E staining, tissues were fixed in 10% zinc formalin, and staining and scanning were performed by the SBP histology core facility.
For H&E staining, tissues were fixed in 10% zinc formalin, and staining and scanning were performed by the SBP histology core facility.
4
Comprehensive Cellular Analysis Protocol
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Adenine (A2786), hemin (H9039), d-biotin (B4639), folic acid (F8758), 100x RPMI vitamin mix (R7256), 100x RPMI amino acid mix (R7131), hygromycin B (H3274), phleomycin (P9564), Wright-Giemsa Stain (WG16) and proteinase K (P4250) were acquired from Sigma-Aldrich. M199 with Hanks’ salts (M2852) was acquired from US Biological Life Sciences. L-biopterin (11.203) was acquired from Schircks Laboratories (Switzerland). G418 (04 727 878 001) was acquired from Roche. Peanut Agglutinin (L107025) was acquired from Vector Laboratories. AquaVi-421 LIVE/DEAD fixable cell dye (L34955) and SuperScript IV First Strand Synthesis System (18091050) were acquired from Life Technologies. Ambion PureLink RNA mini kit (12183025), random primers (48190011), TRIzol Reagent (15596018), and PureLink DNase (12185010) were acquired from Life Technologies.
5
Isolation and Intradermal Infection of Leishmania major Metacyclics
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L. major clone V1 (MHOM/IL/80/Friedlin) promastigotes were grown as previously described57 . Briefly, infectious-stage metacyclic promastigotes of L. major were isolated from stationary cultures (4–5 days old) by negative selection using peanut agglutinin (Vector Laboratories). Mice were infected 4 weeks after boost by injecting 750 metacyclic promastigotes intradermally within both ears. Personnel blinded to the vaccination assignments of the infected mice recorded weekly ear measurements using a metric calliper.
6
Analyzing MALT1 Activity in Spleens
7
Leishmania Promastigote Culture and Isolation
8
Lectin and Selectin Binding Assay
9
Immunofluorescence Staining of Frozen Tissue
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Organs were fixed in 10% neutral buffered formalin, and 3-µm sections were stained with H&E. Immunofluorescence staining was performed on 5–7-µm optical cutting temperature compound–embedded frozen tissue sections. After acetone fixation, sections were incubated with 10% normal goat serum to block nonspecific binding of Ig. Primary staining was performed using rat anti–mouse B220 (RA3-6B2; eBioscience), IL-6 (MP5-20F3; Bio X Cell), or peanut agglutinin (Vector Laboratories). Bound biotinylated Abs or lectin was detected by staining with Texas red–conjugated Streptavidin (Invitrogen) or Alexa Fluor 488–conjugated goat anti–rat IgG(H+L) Abs (Molecular Probes). Bright-field and fluorescence images were captured on a microscope (BX51 or IX51; Olympus) equipped with a camera (NP70; Olympus). Image analysis was performed using ImageJ software (National Institutes of Health).
10
Immunohistochemical Analysis of Splenic Architecture
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