Subcellular Localization of LmBrf1 Protein

The subcellular localization of LmBrf1 labeled with a PTP tag was determined by indirect immunofluorescence, as previously described [29 (link),39 (link)]. Parasites fixed with paraformaldehyde were stained for double indirect immunofluorescence analysis using a mixture of primary antibodies against Prot C (Delta Biolabs, Gilroy, CA, USA) (to detect LmBrf1-PTP) and LmNop56, followed by anti-rabbit immunoglobulin G (IgG) conjugated with Alexa Fluor 568 (red) and anti-mouse IgG coupled with Alexa Fluor 488 (green) secondary antibodies. DNA was stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Individual optical sections were obtained with a Nikon A1R+ STORM confocal microscope, analyzed, and prepared for presentation using the ZEN 2012 software (Blue edition).

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Florencio-Martínez L.E., Cano-Santiago A., Mondragón-Rosas F., Gómez-García M., Flores-Pérez C., Román-Carraro F.C., Barocio-Rodríguez L.A., Manning-Cela R.G., Nepomuceno-Mejía T, & Martínez-Calvillo S. (2021). Participation of TFIIIB Subunit Brf1 in Transcription Regulation in the Human Pathogen Leishmania major. Genes, 12(2), 280.

Publication 2021

A1R+ STORM confocal microscope

Alexa 568 Alexa fluor 488 Anti immunoglobulin Antibodies Confocal microscope Immunoglobulin g Indirect immunofluorescence Mouse Paraformaldehyde Parasites Rabbit Sections

Corresponding Organization : Universidad Nacional Autónoma de México

Other organizations : Instituto Politécnico Nacional, Center for Research and Advanced Studies of the National Polytechnic Institute

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Variable analysis

independent variables

Subcellular localization of LmBrf1 labeled with a PTP tag

dependent variables

Immunofluorescence staining patterns of LmBrf1-PTP and LmNop56

control variables

Parasite fixation with paraformaldehyde
Primary antibodies against Prot C (to detect LmBrf1-PTP) and LmNop56
Secondary antibodies (anti-rabbit IgG conjugated with Alexa Fluor 568 and anti-mouse IgG coupled with Alexa Fluor 488)
DNA staining with DAPI

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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