Naïve WT and ICAM-1/2-/- MedLNs were harvested, fixed for 2 hours at RT in 1% PFA/PBS solution and then placed in 30% sucrose solution O.N. at 4°C. The next day, samples were transferred to a fresh solution of 30% sucrose for an additional 24 hours at 4°C. On the third day, each MedLN was placed in a small cryomold (Fisher scientific, Cat. NC9511236), embedded in OCT compound (Sakura, Cat. 4583), snap frozen on dry ice nuggets and stored at -80°C. Tissue sections (10μm thick) were cut using a cryostat and slides were placed in chilled 70% alcohol for 7 minutes. Prior to staining slides were rehydrated twice with PBS, treated for 5 minutes in 1% SDS/PBS, washed 3 times and blocked with 3% BSA in PBS-T (PBS/0.05% Tween-20). Sections were incubated with primary antibodies diluted in 1% BSA in PBS-T O.N. at 4°C. The following antibodies were used for staining: mouse monoclonal anti-VCAM-1 IgG (clone 429, BD Bioscience, Cat. 550547) and mouse monoclonal anti-PNAd IgM (MECA-79, Biolegend, Cat. 120802). After washing 3 times for 5 minutes in PBS-T at room temperature, the sections were incubated at 4°C O.N. with the following secondary antibodies: anti-rat IgG conjugated to Dylight 549 (Jackson, Cat. 712-505-153) and anti-rat IgM conjugated to Alexa Fluor 647 (Jackson, Cat. 716-605-020). The next day slides were washed and stained with DAPI (1:3000) (Biolegend, Cat. 422801).
Meca 79
Manufactured by BioLegend
Sourced in United States
MECA-79 is a monoclonal antibody that targets a specific antigen. It is designed for use in laboratory research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd62l mel 14, by American Type Culture Collection (2 mentions)
Anti cd11a m17 4, by BioXCell (2 mentions)
Anti ccr7 4b12, by Thermo Fisher Scientific (2 mentions)
Meca 367, by BioXCell (2 mentions)
Celltrace violet ctv, by Thermo Fisher Scientific (2 mentions)
Pertussis toxin ptx, by Merck Group (2 mentions)
Magnetic bead, by Miltenyi Biotec (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by BioLegend (1 mentions)
Anti collagen 4, by Abcam (1 mentions)
Anti collagen 1, by Abcam (1 mentions)
W6 32, by BioLegend (1 mentions)
Anti caspase 3 4 1 18, by BioLegend (1 mentions)
Anti mouse human ki 67 11f6, by BioLegend (1 mentions)
Anti alpha smooth muscle actin α sma, by Abcam (1 mentions)
Anti fibronectin, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Envision flex system, by Agilent Technologies (1 mentions)
Pretreatment module, by Agilent Technologies (1 mentions)
Spectral dapi, by Akoya Biosciences (1 mentions)
Clone c8 144b, by Agilent Technologies (1 mentions)
11 protocols using meca 79
1
Immunofluorescence Staining of Mediastinal Lymph Nodes
2
Comprehensive Immunohistochemical Analysis of PDAC Tumor Microenvironment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen OCT blocks of tumors were cut using a cryostat into 8-μm thick sections and were stained using anti-PNAd (MECA79), anti-human CD31 (WM59, BioLegend), anti-Caspase-3 (4-1-18, BioLegend), anti-Collagen I (abcam), anti-Collagen IV (abcam), anti-Fibronectin (abcam), anti-alpha smooth muscle Actin (α-SMA, abcam), HECA 452 (HECA-452, BioLegend), anti-human HLA-A,B,C antibody (W6/32, BioLegend) and anti-mouse/human Ki-67 (11F6, BioLegend) antibodies. DAPI (VECTASHIELD, Vector Laboratories Burlingame, CA) was used to stain the cell nuclei. The stained tissue sections were imaged using a fluorescent confocal microscope and an EVOS FL2 auto microscope. For immunohistochemistry (IHC) staining, the human post-mortem PDAC samples were stained with anti-mouse/human PNAd (MECA79).
3
Immunohistochemical Characterization of Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formalin-fixed, paraffin-embedded sections were immunostained for CD3 (ready-to-use (RTU) rabbit polyclonal anti-human antibodies; Dako, Glostrup, Denmark), CD4, CD8, CD20, CD68, CD31 (RTU mouse monoclonal anti-human antibodies; Dako) and peripheral node addressin (PNAd) clone MECA-79 (1/3000; Biolegend, San Diego, CA, USA). All immunostains were performed on 5-μm-thick paraffin sections using the Dako autostainer. For visualisation, the Envision Flex system (Dako) was used and antigen retrieval was carried out using the Dako pretreatment module at pH=6. For PNAd, an additional incubation of 30 min was necessary with a rabbit anti-rat immunoglobuline (1/75 +10% normal human serum, Dako).
4
Trafficking of Naïve T-Cells in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Naïve OT-I T-cells isolated from pooled LN and spleen of OT-I RAG1−/−/Thy1.1 mice (4×106 cells) or polyclonal CD8+ T-cells pooled from LN and spleen of C57BL/6 mice and depleted of CD44 expressing cells by magnetic beads (Miltenyi) were injected into the lateral tail vein of recipients. In blocking experiments, cells were incubated with 100 ng/mL pertussis toxin (PTX, Sigma) for 1 h at 37°C, or 100 µg of rat IgG (Jackson Immunoresearch), anti-CD62L (Mel-14, ATCC), or anti-CD11a (M17/4, BioXcell), or 50 µg of anti-CCR7 (4B12, eBioscience) and washed before injection. Luminal PNAd or MAdCAM-1 were blocked by injection of 100 µg of anti-MAdCAM-1 (MECA-367, BioXcell), anti-PNAd (MECA-79, BioLegend) or isotype rat IgG or IgM i.v. 1 h prior to T-cell transfer. In some experiments, cells were labeled with Cell Trace Violet (CTV) (Invitrogen) prior to transfer. Tissues were harvested 1 h or 18 h after naïve T-cell transfer.
5
Multiplex Immunohistochemical Analysis of Tertiary Lymphoid Structures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AR9, CD8 (1:500, clone C8/144B Agilent Technologies, Santa Clara, California, USA) Opal540; AR6, CD20 (1:1000, clone L26, Agilent Technologies) Opal520; AR6, PNAd (1:1000 clone MECA-79, BioLegend, San Diego, California, USA) Opal620; AR6, Ki67 (1:20, SP6, Abcam, Cambridge, Massachusetts, USA) Opal690; and AR6, spectral DAPI (Akoya Biosciences, Marlborough, Massachusetts, USA). A representative image from panel 1 provided for a TLSpos lesion (online supplemental figure 2 ) and a TLSneg lesion (online supplemental figure 3 ).
6
Multicolor Immunofluorescence Imaging of Liver
7
Trafficking of Naïve T-Cells in Mice
8
Comprehensive Immune Profile of HGSOC Metastases
9
Multicolor Immunofluorescence Staining of Organs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The organs were fixed in 1% formaldehyde–10 mM sodium periodate–75 mM l -lysine (18 h, 4°C), equilibrated in 30% sucrose (18 h, 4°C), and then frozen. Sections were blocked with 0.1% Triton X-100–5% normal goat serum and then incubated (18 h, 4°C) with antibodies to B220 (rat MAb RA3-6B2), surfactant protein C (SPC) (goat polyclonal antibody [pAb]), CK19 (goat MAb N-13; Santa Cruz Biotechnology), CD68 (rat MAb FA-11), ER-TR7 (rat MAb), CD31 (rat MAb MEC 7.46), β-galactosidase (chicken pAb), LYVE-1 (rabbit pAb; Abcam), podoplanin (PDP) (goat pAb, R&D Systems), CD11c (hamster MAb HL-3; BD Pharmingen), CD206 (rat MAb MR5D3) and CD169 (rat MAb 3D6.112) (Serotec), peripheral node addressin (PNAd) (rat MAb MECA-79; BioLegend), or aquaporin V (rabbit pAb; Alamone Labs). After incubation with primary antibodies, the sections were washed three times in phosphate-buffered saline (PBS), incubated (1 h, 23°C) with combinations of Alexa Fluor 488-, Alexa Fluor 568-, or Alexa Fluor 647-conjugated goat pAb (Abcam), then washed three times in PBS, stained with 4′,6′-diamidino-2-phenylindole (DAPI), and mounted in ProLong gold (Life Technologies). TdTomato (Tom) and GFP expression were visualized directly. Images were acquired with a Zeiss LSM510 microscope and analyzed with Zen software.
10
Investigating LYVE-1's Role in Group A Streptococcus Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
FVB/n female mice (4–5 weeks old (Charles River, Margate,UK)), C57Bl/6 and C57Bl/6 LYVE-1-/- female mice (originally obtained from Regeneron Pharmaceuticals, USA) were challenged intra-muscularly with 1×108 GAS, and quantitative endpoints compared at 3 or 24 hours post infection. For LYVE-1 blocking experiments mice were injected intra-peritoneally with 0.5 mg anti mLYVE-1 mAb2125 (R&D) or control rat IgG (isotype control or polyclonal) (R&D) 24 hours prior to infection. Mice were euthanized, blood taken by cardiac puncture and infected thigh muscle, spleen, liver, and left and right inguinal lymph nodes dissected. All organs were plated to quantify bacterial cfu and systemic dissemination. For imaging of draining inguinal lymph nodes, wildtype C57Bl/6 and C57Bl/6 LYVE-1-/- mice (n = 3) were challenged intra-muscularly in both thighs with 1×108 GAS. Three hours post infection lymph nodes were dissected and processed as described above for draining cervical lymph nodes, then stained with anti-mouse LYVE-1 (mAb C1/8), FITC-conjugated group A carbohydrate antibody (Abcam), anti-mouse podoplanin (eBioscience, clone eBio8.1.1) or anti-mouse PNAd (Biolegend, clone MECA79).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!