To visualize TFH and TFR cells in lymph node tissues, immunofluorescence staining was conducted according to a previously described method (38 (link)). Goat polyclonal anti-human PD-1 Abs (catalog number AF1086, 1:100; R&D Systems), rabbit monoclonal anti-human CD4 Abs (clone EPR6855, 1:200; Abcam), and mouse monoclonal anti-human Foxp3 Abs (clone 236A/E, 1:200; Abcam) were incubated with tissues sections. Following primary Ab incubation and washing, donkey anti-goat IgG conjugated with Alexa Fluor 488 (catalog number A-11055, 1:100), donkey anti-rabbit IgG conjugated with Alexa Fluor 594 (catalog number R37119, 1:100), and donkey anti-mouse IgG conjugated with Alexa Fluor 647 (catalog number A-31571, 1:100; all from Thermo Fisher Scientific) were used. Cell nuclei were counterstained with DAPI. A Nikon A1R-TiE live cell imaging confocal system was used to visualize and capture images of stained samples.
Donkey anti rabbit igg conjugated with alexa fluor 594
Manufactured by Thermo Fisher Scientific
Donkey anti-rabbit IgG conjugated with Alexa Fluor 594 is a secondary antibody used in various immunodetection techniques. It binds to rabbit primary antibodies and is labeled with the Alexa Fluor 594 fluorescent dye, which has an excitation/emission maxima of 590/617 nm.
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Lab products found in correlation
Donkey anti goat igg conjugated with alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
A1r tie live cell imaging confocal system, by Nikon (1 mentions)
Eclipse ti u inverted, by Nikon (1 mentions)
C2 microscope, by Nikon (1 mentions)
Cryotome fse, by Thermo Fisher Scientific (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
2 protocols using donkey anti rabbit igg conjugated with alexa fluor 594
1
Visualizing T cells in Lymph Nodes
2
Cryosectioning and Immunostaining of Mouse Limb Muscles
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The mouse limb muscles with bioluminescence signal were dissected and frozen in O.C.T compound (Sakura Finetek USA, Torrance, California) on dry ice and then placed into the −80°C freezer for a minimum of 24 hours. The tissue was then sectioned using a Cryotome FSE (Thermo Scientific, Waltham, Massachusetts) set to 10–15 μm. Immunostaining was performed as we previously described.35 The goat anti‐GFP antibodies or rabbit anti‐GFP antibodies (Novus Biologicals, Littleton, Colorado), rabbit anti‐human CD31 antibodies (Bethyl Laboratories, Montgomery, Texas), and sheep anti‐FVIII antibodies (Affinity Biologicals, Ancaster, Canada) were diluted in PBS with 1% bovine serum albumin (BSA) to 100, 200, and 50 folds, respectively. Secondary antibodies including donkey anti‐goat IgG conjugated with AlexaFluor 488 (ThermoFisher Scientific), donkey anti‐rabbit IgG conjugated with AlexaFluor 594 (ThermoFisher Scientific), and donkey anti‐sheep IgG conjugated with AlexaFluor 647 (EMD Millipore, Burlington, Massachusetts) were diluted to 500 folds in PBS with 1% BSA, respectively. Fluorescence images were captured using a Nikon Eclipse Ti‐U Inverted or Nikon C2 microscope (Nikon Instruments Inc, Melville, New York).
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