NHS cryosections of 5 µm thickness were blocked with TBS-Ca2+ (Bio-Rad) plus 1% BSA (Sigma-Aldrich) at RT for 30 min. Slides were washed with TBS-Ca2+ three times and incubated with samples diluted in TBS-Ca2+ plus 1% BSA at RT for 60 minutes. Binding of scFvs was detected through staining with rat anti-HA mAb (3F10; dilution, 1:100; Roche), followed by an Alexa Fluor 594-conjugated anti-rat IgG (dilution, 1:200; Invitrogen). Analogously, IIF was performed on ME substrates (SCIMEDX; EUROIMMUN), following above protocol.
Alexa fluor 594 conjugated anti rat igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 594-conjugated anti-rat IgG is a secondary antibody reagent that binds to rat immunoglobulin G (IgG) and is labeled with the Alexa Fluor 594 fluorescent dye. It is designed for use in various immunochemical techniques, such as immunofluorescence microscopy, flow cytometry, and Western blotting, where the detection of rat IgG is required.
Automatically generated - may contain errors
Lab products found in correlation
Scimedx, by EUROIMMUN (1 mentions)
Tbs ca2, by Bio-Rad (1 mentions)
Rat anti ha mab 3f10, by Roche (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Alexa fluor 568 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated anti gfp, by Thermo Fisher Scientific (1 mentions)
Tcs sp8x white light laser confocal system, by Leica (1 mentions)
Pdgfrβ, by Abcam (1 mentions)
α sma, by Abcam (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
4 protocols using alexa fluor 594 conjugated anti rat igg
1
Immunohistochemical Staining of scFvs
2
Immunofluorescence Staining of Lymphoid Tissues
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LNs, spleens and small intestines containing PPs were harvested, washed with PBS, fixed in 4% PFA for 1 hour and snap-frozen in an OCT-filled mold on a liquid nitrogen-cooled metal surface. Cryosections 10 μm were rehydrated in wash buffer (0.1% saponin in PBS) for 5 min and blocked in PBS containing 0.5% albumin for 30 min. Sections were incubated with antibodies against: collagen IV and ER-TR7 from Abcam; TRANCE, eFluor 660-conjugated anti-podoplanin and anti-Lyve1 from eBioscience; CXCL13 and CCL21 from R&D Systems; CD31 and CD35 from BD Pharmigen; MAdCAM-1, Alexa Fluor 594-conjugated anti-CD3 and Alexa Fluor 647-conjugated anti-B220 from Biolegend; and Alexa Fluor 488-conjugated anti-GFP from Invitrogen. Unconjugated antibodies were detected with the following secondary antibodies: Alexa Fluor 594-conjugated anti-rat-IgG, Alexa Fluor 568-conjugated anti-rabbit-IgG all purchased from Invitrogen. TRANCE, CXCL13 and CCL21 expression were detected using the tiramide amplification signal kit (Applied biosystem). Finally, images were acquired with a TCS SP8X White Light Laser confocal system (Leica).
3
Pericyte Identification in Kidney Sections
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Formalin‐fixed kidney sections underwent deparaffinization, heat‐mediated antigen retrieval in citrate buffer, and nonspecific background blocking. Pericytes were identified by the co‐expression of platelet‐derived growth factor receptor‐β (PDGFR‐β) (Abcam, Cambridge, UK; 1:100) and neural‐glial antigen 2 (NG2) (Abcam, Cambridge, UK; 1:200) antigens, followed by incubation with Alexa Fluor 488‐conjugated anti‐rabbit IgG (Thermo Fisher Scientific, MA, USA) and Alexa Fluor 405‐conjugated anti‐mouse IgG (Thermo Fisher Scientific, MA, USA). Perivascular pericytes were identified and evaluated by co‐staining for vascular endothelial cell markers. The vascular endothelial cell marker cluster of differentiation 31 (CD31) (Dianova, Hamburg, Germany; 1:50) antibody was used in combination with Alexa Fluor 594‐conjugated anti‐rat IgG (Thermo Fisher Scientific, MA, USA). In addition, pericyte‐derived myofibroblasts were identified through co‐staining with α‐SMA (Abcam, Cambridge, UK; 1:100). After incubation with α‐SMA antibody, labeled with Alexa Fluor 594‐conjugated anti‐goat IgG (Thermo Fisher Scientific, MA, USA).
4
Immunohistochemical Analysis of Graft Rejection
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The mice were anesthetized, and the grafts were isolated. The base of the heart grafts was embedded in optimal cutting temperature compound (Sakura Finetechnical) and frozen in liquid nitrogen. Fresh-frozen tissue sections (5-μm thick) were prepared and fixed with acetone. After immersion in phosphate-buffered saline (PBS), the sections were stained with antibodies against Ki67 (718071, clone sp6, ready-to-use, Nichirei Biosciences, Japan), B220 (103202, clone RA3-6B2, dilution 1:100, BioLegend, Japan), CD31 (102432, clone 390, dilution 1:100, BioLegend), Alexa Fluor 480-conjugated IgM (ab150121, dilution 1:200, Abcam), and Alexa Fluor 480-conjugated IgG (a-21202, dilution 1:200, Thermo Fisher Scientific, Waltham, MA, USA). Paraffin sections were stained with anti-mouse CD4 antibody (ab237722, clone CAL4, dilution 1:100, Abcam) and Foxp3 (14-5773-82, clone FJK-16s, dilution 1:100, eBioscience, San Diego, CA, USA), followed by staining with Alexa Fluor 488-conjugated anti-rabbit IgG antibody (A21202, dilution 1:200, Thermo Fisher Scientific) and Alexa Fluor 594-conjugated anti-rat IgG (A21209, dilution 1:200, Thermo Fisher Scientific). Images of the transplanted mice stained with antibodies were acquired using a fluorescence microscope (Olympus BX53, Tokyo, Japan).
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