Journal: STAR Protocols

Article Title: Protocol for investigating tertiary lymphoid structures in human and murine fixed tissue sections using Opal™-TSA multiplex immunohistochemistry

doi: 10.1016/j.xpro.2022.101961

Figure Lengend Snippet: Detailed staining conditions and order of the Opal™-TSA mIHC 6-marker panel for formalin free zinc salt-fixed tissue sections

Article Snippet: Rat monoclonal anti-mouse PNAd [MECA-79] antibody , BD Pharmingen , Cat#553863 (1:5000 dilution).

Techniques: Staining, Incubation

Journal: STAR Protocols

Article Title: Protocol for investigating tertiary lymphoid structures in human and murine fixed tissue sections using Opal™-TSA multiplex immunohistochemistry

doi: 10.1016/j.xpro.2022.101961

Figure Lengend Snippet:

Article Snippet: Rat monoclonal anti-mouse PNAd [MECA-79] antibody , BD Pharmingen , Cat#553863 (1:5000 dilution).

Techniques: Plasmid Preparation, Recombinant, Software, Staining, Imaging

Journal: The American Journal of Pathology

Article Title: Preferential Lymphatic Growth in Bronchus-Associated Lymphoid Tissue in Sustained Lung Inflammation

doi: 10.1016/j.ajpath.2014.01.021

Figure Lengend Snippet: Lymphatics in BALT. A: Montage of H&E-stained sections of mouse left lung at 0, 7, 14, and 28 days after M. pulmonis infection. At 7 days, BALT (intense purple) is greatest in the hilum. The overall amount of BALT increases during the infection. By 28 days, the entire lung consists of regions of BALT surrounded by consolidated lung parenchyma typical of lobar pneumonia (less intense purple areas). B: After 28 days of infection, the main bronchus (Br) and pulmonary artery (PA) are surrounded by BALT, which has a sharp border with the lung parenchyma. The pulmonary vein (PV) has a separate cuff of BALT. Lymphoid follicles (arrows). C: Time course of increase in amount of BALT expressed as percentage of total area of H&E-stained sections of mouse left lung at 0, 7, 14, and 28 days after infection. Infected values are significantly greater than pathogen free and increase with infection duration (∗P < 0.05; N ≥ 5 mice per group). D: Time course of increase in left lung wet weight, expressed relative to the control (0 days) weight, at 0, 7, 14, and 28 days after infection. Infected values are significantly different from pathogen free; values at 28 days are different from values at 7 and 14 days (∗P < 0.05; N ≥ 13 mice per group). E–M: A 28-day infection. E: MECA-79 immunoreactivity (red) marks HEVs (arrowheads) in BALT around bronchus (Br) and pulmonary vein (PA). Blood vessels marked by PECAM-1 immunoreactivity (green). F: M cells stained by U. europaeus lectin (red) in the bronchial epithelium after 28 days of infection. Epithelial cells (pan-keratin, blue). Lymphatics (Prox1-EGFP, green, arrow). G: B-cell follicles (B220, red) and lymphatics (Prox1-EGFP, green) in BALT after infection for 28 days. H: Enlargement of B-cell follicle in G (arrow). B cells (B220, red) are more abundant than T cells (CD3e, cyan). Lymphatics (Prox1-EGFP, green, arrow) in the T-cell zone but not in the B-cell zone. I: Enlargement of perifollicular lymphatic (Prox1-EGFP, green) in H (arrow), showing B cells (B220, red) and T cells (CD3e, cyan) in the lumen. J: Macrophages and dendritic cells (Aif1/Iba1, red) surround lymphatics (Prox1-EGFP, green) and outline the border of BALT around a Br. K: Neutrophils (S100A8, red) scattered near lymphatics (Prox1-EGFP, green) in BALT around a Br and found in clumps (arrow) in the airway lumen. L: Lung parenchyma near pleura showing abundant neutrophils (S100A8, red) and scattered lymphatics (Prox1-EGFP, green). M: Higher magnification of region in L showing neutrophils (S100A8, cyan) and macrophages and dendritic cells (Aif1/Iba1, red) near lymphatics (Prox1-EGFP, green). Scale bars: 1000 μm (A); 400 μm (B and G); 200 μm (E and J–L); 50 μm (F, H, and M); 20 μm (I).

Article Snippet: 17 We used the following antibodies to identify blood vessels: platelet endothelial cell adhesion molecule (PECAM)-1 (CD31) (hamster anti-mouse monoclonal, clone 2H8, MA3105; Thermo Fisher Scientific, Rockford, IL); lymphatics, such as VEGFR-3 (goat anti-mouse polyclonal AF743; R&D Systems, Minneapolis, MN); lymphatic vessel endothelial cell marker (LYVE)-1 (rabbit anti-mouse polyclonal 11-034; AngioBio, Del Mar, CA); podoplanin (Syrian hamster anti-mouse monoclonal, clone 8.1.1; AngioBio); neuropilin-2 (goat polyclonal AF567; R&D Systems); chemokine ligand (CCL) 21 (goat polyclonal AF457; R&D Systems); VEGF-C (goat polyclonal sc-7132; Santa Cruz Biotechnology, Dallas, TX); Prox1 (rabbit anti-mouse polyclonal AB5475; Millipore, Billerica, MA); smooth muscle cells, such as α-smooth muscle actin (αSMA; mouse monoclonal, clone 1A4 conjugated to fluorescein isothiocyanate, F3777, or Cy3; C6198; Sigma-Aldrich); pan-leukocyte marker CD45 (rat anti-mouse monoclonal, clone 30-F11; BioLegend, San Diego, CA); B lymphocytes, such as B220 (rat anti-mouse monoclonal, clone RA3-6B2; BD Biosciences, San Jose CA); T lymphocytes, such as CD3e (Armenian hamster anti-mouse monoclonal, clone 145-2C11; BD Biosciences); macrophages and dendritic cells, such as allograft inflammatory factor 1/ionized calcium-binding protein 1 (Aif1/Iba1; Wako USA, Richmond, VA); neutrophils, such as S100A8 (goat polyclonal AF3059; R&D Systems); high endothelial venules (HEVs), such as MECA-79 (PNAd carbohydrate epitope, CD62L, rat anti-mouse monoclonal, clone MECA-79; BD Biosciences); lung epithelial cells (primarily alveolar type 1 cells), such as aquaporin-5 (rabbit polyclonal ab78486; Abcam, Cambridge, MA) or pan-cytokeratin (rabbit polyclonal Z0622; Dako USA, Carpinteria, CA); epithelial microfold cells (M cells), such as biotinylated Ulex europaeus lectin B1065 (Vector Laboratories, Burlingame, CA); and endogenous EGFP fluorescence, amplified by chicken polyclonal anti-GFP (GFP-1020; Aves Labs, Tigard, OR).

Techniques: Staining, Infection