C57/BL6 mice (2 males and 2 females, 10 weeks) were injected intracardially with 0.75 mg 10kD Alexa Fluor™ 568 dextran (Invitrogen, #D22912) and sacrificed after 3 min (using isoflurane). Kidneys were harvested and immediately sectioned. Dextran was visualized in living kidney sections (2 mice). The remaining kidney was fixed and sectioned, and stained with antibodies to highlight PT segments (S1: SGLT2, Abcam, #85626, 1:400; S2: OAT1, Alpha Diagnostic, #OAT11A, 1:200; S3: aquaporin-4, Alomone Labs, #AQP-004, 1:500). Images were obtained using a Leica SP8 inverted confocal microscope with environmental chamber, motorized stage, and 25X 0.95 numerical aperture (NA) and 40X 1.1 NA water objectives. Tilescans were generated to document important anatomic landmarks to facilitate interpretation of PT segments. Dextran uptake was grossly quantitated from line profiles drawn perpendicular to the PT segment using LASX software from maximum projections of the live dataset (3 tilescans from one male). Approximately 45 line scans contributed to mean intensity estimate for presumed S2 and ten line scans for confirmed S1 as it was observed to emerge from glomerulus.
Alexa fluor 568 dextran
Manufactured by Thermo Fisher Scientific
Alexa Fluor 568 dextran is a fluorescent dye conjugated to dextran, a branched polysaccharide. It can be used as a tracer or marker in biological applications.
Automatically generated - may contain errors
3 protocols using alexa fluor 568 dextran
1
Quantifying Dextran Uptake in Kidney PT Segments
2
Knockdown and Rescue of Hspb1 in Xenopus
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Wild-type X. tropicalis adults (male and female) were purchased from the NASCO. Methods involving live animals were carried out in accordance with the guidelines and regulations approved and enforced by the Institutional Animal Care and Use Committees at the University of Delaware. Morpholinos (MOs) were designed using Gene Tools, LLC, OR. The Hspb1 MO is a 25-mer with the sequence 5′ GTA TTC TGC GTT CTG ACA TTT TC 3′. The control MO is the standard control MO obtained from Gene Tools with the sequence 5′ CCT CTT ACC TCA GTT ACA ATT TAT A 3′. Embryos were collected and injected with a PLI-100A microinjector (Harvard Apparatus) as described previously (106 (link)). Control or HSPB1 morpholino (1.5 ng per blastomere) was injected into a single dorsal-animal blastomere at the eight-cell stage; Alexa Fluor 568 dextran (Invitrogen #D22912) was co-injected as a lineage tracer. For the rescue experiments, mouse Hspb1 mRNA was generated by in vitro transcription as described (107 (link)) and co-injected with the HSPB1 morpholino (50 pg mRNA per blastomere). Injected embryos were cultured in 0.1X MBS to desired stages, and eye phenotypes were observed using a Zeiss Axiozoom.v16 epifluorescence microscope.
3
Dye-Filling of Neurons in Caudal NM
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Neurons in the caudal NM were individually dye-filled using electroporation (Wang and Rubel, 2012 (link)). Briefly, a glass pipette filled with fixable Alexa Fluor 568 dextran (Invitrogen) was driven to approach an identifiable cell body under a Zeiss V16 stereo-fluorescence microscope. The dye was introduced into the cell by a positive voltage (15–30 V, 20-ms pulse duration, 20 pulses/s, 1–5 s). After electroporation, slices were incubated for another 1–2 min to allow dye diffusion to distal dendrites. Slices were then fixed with 4% paraformaldehyde for 15 min at room temperature. After washing with PBS, sections were counterstained with NeuroTrace and mounted on noncoated slides with Fluomount mounting medium. To reduce tissue shrinkage, a nail polish spot was made at each corner of the coverslip to increase the space between the slide and coverslip.
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