Rabbit anti trfp

Manufactured by Evrogen

Rabbit anti-tRFP is a polyclonal antibody raised against the tRFP (tandem Red Fluorescent Protein) reporter protein. It is used for the detection and localization of tRFP-tagged proteins in various applications, such as Western blotting, immunocytochemistry, and immunohistochemistry.

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4 protocols using rabbit anti trfp

Immunostaining of Hcrt and Cxcr4b in Zebrafish

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All groups of 20, 24 and 28 hpf Hcrt × Cxcr4b zebrafish embryos were fixed overnight at 4 °C in 4% PFA in PBST (1% Tween-20) as previously described^{39 (link)}. Samples were washed 4 × 15 min with 1% PBST and blocked for 2 h in PBS containing 2% normal donkey serum, 0.5% Tween-20, and 1% BSA. The samples were incubated overnight at 4 °C with shaking in chicken anti-GFP (Aves Labs; 1:500) and rabbit anti-tRFP (Evrogen; 1:500) primary antibodies diluted with the blocking solution. The samples were washed 4 × 15 min with 0.1% PBST and incubated overnight at 4 °C with shaking in Alexa fluor 488 anti-chicken (Invitrogen; 1:500) and Alexa Fluor 647 anti-rabbit (Abcam; 1:500) secondary antibodies and DAPI (1:200) diluted in 0.2% PBST. Lastly, the samples were washed 4 × 15 min with 0.2% PBST and stored at 4 °C.

Collier A.D., Yasmin N., Karatayev O., Abdulai A.R., Yu B., Khalizova N., Fam M, & Leibowitz S.F. (2023). Neuronal chemokine concentration gradients mediate effects of embryonic ethanol exposure on ectopic hypocretin/orexin neurons and behavior in zebrafish. Scientific Reports, 13, 1447.

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Immunofluorescence Imaging of Drosophila Fat Body

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Fat body tissues from 68 to 85 hours AEL larvae were dissected in PBS with 2% formaldehyde at room temperature, fixed 20 to 30 min in 4% formaldehyde, washed twice for 10 min each in PBST 0.3%, blocked for 30 min (PBST, 5% bovine serum albumin (BSA), 2% fetal bovine serum, 0.02% NaN₃), incubated with primary antibodies in the blocking buffer overnight, and washed four times for 15 min each. Secondary antibodies diluted 1:200 or 1:500 in PBST were added for 1 hour and tissues were washed four times before mounting in Vectashield/ 4′,6-diamidino-2-phenylindole (DAPI). Rabbit anti P-4EBP1 from Cell Signaling Technologies (CST 236B4, #2855) was diluted 1:500, rabbit anti-tRFP was from Evrogen (#AB233) and was used against mKate2 to stain dCTNS-mKate2. Purified polyclonal antibody against Mitf was generated in guinea pig by the company Eurogentec using the epitope: CRRFNINDRIKELGTL. Samples were imaged using Zeiss LSM 780 and Leica TCS SP8 SMD confocal systems with a 40x water or oil immersion objective, and images were processed with Fiji software.

Jouandin P., Marelja Z., Shih Y.H., Parkhitko A.A., Dambowsky M., Asara J.M., Nemazanyy I., Dibble C.C., Simons M, & Perrimon N. (2022). Lysosomal cystine mobilization shapes the response of TORC1 and tissue growth to fasting. Science (New York, N.Y.), 375(6582), eabc4203.

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Immunofluorescent Staining of Embryos

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Antibody staining was performed as previously described^{72 (link)}. The antibody against activated leukocyte cell adhesion molecule (Alcam) was used at a 1:25 dilution in PBS with 4% bovine serum albumin and 0.3% Triton (PBT). Rabbit anti-tRFP was obtained from Evrogen and used at 1:200 dilution. Secondary goat anti-mouse antibodies and goat anti-rabbit antibodies (Alexa 633, Alexa 568; Invitrogen) were used at 1:200 dilution in PBT. Embryos were mounted in Vectashield with DAPI (Vector Laboratories).

Gawdzik J.C., Yue M.S., Martin N.R., Elemans L.M., Lanham K.A., Heideman W., Rezendes R., Baker T.R., Taylor M.R, & Plavicki J.S. (2018). sox9b is required in cardiomyocytes for cardiac morphogenesis and function. Scientific Reports, 8, 13906.

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Multicolor Immunostaining of Embryos

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Embryos were fixed in 4% paraformaldehyde (PFA) for 8-12 hr. Whole-mount staining was carried out according to previously described protocols (Norden et al., 2009) . Primary Antibodies Mouse anti-g-tubulin (Sigma-Aldrich; 1:250), rat anti-pH3 (Abcam; 1:500), rabbit anti-pH3 (Chemicon, 1:500), rabbit anti-tRFP (Evrogen, 1:500), and mouse anti-ZO1 (Invitrogen 1:200) were used. Secondary Antibodies Alexa Fluor 488 donkey antirabbit, Alexa Fluor 488 chicken antimouse, Alexa Fluor 568 goat antirat, Alexa Fluor 594 goat antirabbit, Alexa Fluor 647 goat antirat, Alexa Fluor 647 goat antirabbit, Alexa Fluor 647 goat antimouse (all Molecular Probes, all 1:1,000) were used.
Additionally, for detection of GFP and RFP fluorophores, GFP booster and RFP booster (ChromoTech) were used (1:400 dilution). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (1:5,000).

Strzyz P.J., Lee H.O., Sidhaye J., Weber I.P., Leung L.C, & Norden C. (2015). Interkinetic nuclear migration is centrosome independent and ensures apical cell division to maintain tissue integrity. Developmental cell, 32(2).

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