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Af488 donkey anti rat

Manufactured by Thermo Fisher Scientific
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AF488 donkey anti-rat is a secondary antibody conjugated with Alexa Fluor 488 dye. It is designed to detect and bind to primary antibodies raised in rat. This product is commonly used in various immunoassay techniques, such as immunofluorescence and flow cytometry, to visualize and identify rat-specific target proteins or cells.

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Lab products found in correlation

Af594 goat anti mouse, by Thermo Fisher Scientific (2 mentions) Lsm 880, by Zeiss (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Qwin pro, by Leica (1 mentions) Rabbit anti cd20, by Abcam (1 mentions) Rf8b2, by BD (1 mentions) Dm5000b fluorescent microscope, by Leica (1 mentions) Rpa t4, by BD (1 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) Anti ptn, by Santa Cruz Biotechnology (1 mentions) Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions) Anti aqp7, by Novus Biologicals (1 mentions) Af594 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions) Anti rage, by Abcam (1 mentions) Zen software, by Zeiss (1 mentions) Anti cd31, by BD (1 mentions) Fluorescence mounting medium, by Agilent Technologies (1 mentions) Goat anti rabbit af488, by Thermo Fisher Scientific (1 mentions) Mq1 17h12, by Thermo Fisher Scientific (1 mentions) C8487, by Merck Group (1 mentions)

4 protocols using af488 donkey anti rat

1

Quantifying Lymph Node Immune Cell Subsets

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Frozen lymph node sections 4 μm thick were fixed in 1% paraformaldehyde (Sigma, St. Louis, MO), stained for 1 hour with rat anti-CXCR5 (RF8B2, BD Bioscience), mouse anti-CD4 (RPA-T4, BD Bioscience) and rabbit anti-CD20 (Abcam, Cambridge, MA), then treated for 30 min with secondary antibodies AF488 donkey anti-rat, AF647 chicken anti-rabbit and AF594 goat anti-mouse (Invitrogen, Grand Island, NY). Stained slides were viewed on a Leica DM5000B fluorescent microscope and 10 to 15 randomly selected areas were imaged using Qwin Leica FW4000 software (Leica). Follicular and extrafollicular tissue areas within the images were defined morphologically by CD20 staining, and percentages of CXCR5+, CD4+, and CXCR5+CD4+ tissue area determined by quantitative image analysis (QWin Pro; v.3.4.0, Leica).
Kohler S.L., Pham M.N., Folkvord J.M., Arends T., Miller S.M., Miles B., Meditz A.L., McCarter M., Levy D.N, & Connick E. (2016). Germinal Center T Follicular Helper Cells (GC TFH) are Highly Permissive to HIV-1 and Alter Their Phenotype During Virus Replication. Journal of immunology (Baltimore, Md. : 1950), 196(6), 2711-2722.
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2

Immunofluorescence Staining of Lung, Brain, and Heart Tissues

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The slides were permeabilized and blocked with 10% donkey serum, 2% BSA, 0.05% tween in PBS for 1 hr at room temperature. For lung cells, the slides were incubated with primary antibodies anti-CD31 (BD Pharmingen, Cat#: 550274, 1:25) and anti-RAGE (Abcam, Cat#: Ab3611, 1:3200) at 4°C overnight. The brain ECs were incubated with primary antibodies anti-CD31 (BD Pharmingen, Cat#: 550274, 1:25) and anti-PTN (Santa Cruz Biotechnology, Cat#: sc-74443, 1:3200) at 4°C overnight. For the heart samples, primary antibodies anti-AQP7 (Novus Biologicals, Cat#: NBP1-30862, 1:3200) and anti-CD31 (BD Pharmingen, Cat#: 550274, 1:25) were used and incubated at 4°C overnight. The next day, slides were washed and incubated with the fluorescence-conjugated secondary antibody (AF488 donkey anti-rat 1:300, Invitrogen Cat#: A-21208; AF594 donkey anti-rabbit 1:300, Invitrogen Cat#: A-21207; AF594 goat anti-mouse 1:300, Invitrogen Cat#: A11032), followed by washing with 1x PBS. Cells were stained with DAPI and mounted on ProLong Gold mounting medium (Invitrogen, Cat#: P36934). Images were taken with a confocal microscope LSM880 (Zeiss) and analyzed by Zen software (Zeiss).
Jambusaria A., Hong Z., Zhang L., Srivastava S., Jana A., Toth P.T., Dai Y., Malik A.B, & Rehman J. (2020). Endothelial heterogeneity across distinct vascular beds during homeostasis and inflammation. eLife, 9, e51413.
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3

Multiplex Immunofluorescence Staining of Cryosections

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Cryosections of 10 μm were made of tumors and organs previously frozen in cryoembedding medium. Sections were fixed in ice-cold acetone, blocked with 20 % FBS in 3 % BSA and stained for CD4+ T cells (GK1.5, BioLegend), CD8+ T cells (53-6.7, BioLegend), activated Caspase 3 (C8487, Sigma) and CD31+ endothelial cells (AF36288, R&D systems). Secondary antibodies used were goat-anti-rabbit-AF488, donkey-anti-rat-AF488 and donkey-anti-goat-AF594 (Thermo Fisher Scientific). For ex vivo immunofluorescence studies, FITC labelled IgG was detected using rabbit-anti-FITC (Bio-Rad) and donkey-anti-rabbit-AF488 (Thermo Fisher Scientific) as secondary antibody. XE-hIL2, XE-mIFNα2 and XE-TNF were detected using anti-hIL2 (MQ1-17H12, eBioscience), anti-mIFNα2 (50525-T08, SinoBiological) and anti-mTNF (MP6-XT22, Invitrogen) antibodies, respectively. A rabbit-anti-rat antibody (ab102248, abcam) was used in case of anti-hIL2 and anti-mTNF staining. Detection was performed using the Alexa Fluor™ 488 Tyramide SuperBoost™ Kit with goat-anti-rabbit-IgG (B40943, Thermo Fisher) according to the manufacturers’ instruction. Nuclei were counterstained with DAPI (Thermo Fisher Scientific). Tumor sections were mounted with fluorescence mounting medium (Dako) and analyzed using an Axioscop Mot Plus Microscope (Zeiss).
Ziffels B., Stringhini M., Probst P., Fugmann T., Sturm T, & Neri D. (2019). Antibody-based delivery of cytokine payloads to carbonic anhydrase IX leads to cancer cures in immunocompetent tumor-bearing mice. Molecular cancer therapeutics, 18(9), 1544-1554.
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4

Clec9a mRNA Expression Analysis

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Clec9a mRNA expression was detected using the RNAscope Multiplex Fluorescent v2 assay combined with immunofluorescence (ACD) following manufacturer’s instructions. Lymph nodes or injured spinal cords from PBS and 4% paraformaldehyde (PFA) perfused Clec9a+/CreRosaLSLtdTomato mice were further fixed with 4% PFA (16h, 4C) and cryoprotected with 30% sucrose (16h, 4C) prior to making 10 μm thick frozen sections. Clec9a mRNA was detected using probe mm-Clec9a-01 (537731, ACD) in conjunction with TSA633 (NEL745001, Perkin Elmer). Sections were further stained with a rat anti-CD45 (1:100, clone 30-F11, Biolegend), detected with a donkey anti-rat AF488 (1:400, Thermo Fischer Scientific); and with a rabbit anti-RFP (1:600, polyclonal, Rockland), detected with a donkey anti-rabbit AF555 (1:400, polyclonal, Thermo Fischer Scientific). Nuclei were counterstained with Hoechst. Images were acquired on a LSM880 inverted confocal microscope (Zeiss). Number of Clec9a puncta per cell were quantified using ImageJ/FIJI. Background level of the assay was determined by counting the maximum number of Clec9a puncta in Clec9a-deficient animals (Clec9aCre/CreRosaLSLtdTomato/LSLtdTomato).
Frederico B., Martins I., Chapela D., Gasparrini F., Chakravarty P., Ackels T., Piot C., Almeida B., Carvalho J., Ciccarelli A., Peddie C.J., Rogers N., Briscoe J., Guillemot F., Schaefer A.T., Saúde L, & Reis e Sousa C. (2022). DNGR-1-tracing marks an ependymal cell subset with damage-responsive neural stem cell potential. Developmental Cell, 57(16), 1957-1975.e9.
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