Mice received a tail vein injection of 100 µl of Biocytin-TMR (1% in PBS; ThermoFisher Scientific). The dye was allowed to circulate for 30 minutes. Animals were anesthetized with isoflurane and perfused first with PBS, then with 4% PFA in PBS. AlexaFluor 488-conjugated goat anti-mouse IgG (Invitrogen, 1:500) was used to visualize serum IgG leakage in brain sections.
Biocytin tmr
Manufactured by Thermo Fisher Scientific
Biocytin TMR is a fluorescent dye used for labeling and tracking biomolecules. It consists of biotin conjugated to the rhodamine dye tetramethylrhodamine (TMR). Biocytin TMR can be used to label and visualize proteins, nucleic acids, and other biomolecules in various applications.
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5 protocols using biocytin tmr
1
Tracking Vascular Permeability in Mice
2
Biocytin-TMR Retina and Liver Labeling
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Mice were anesthetized using isoflurane and injected with 1% Biocytin-5(and-6-)-Tetramethylrhodamine (Biocytin-TMR: ThermoFisher Scientific; Waltham, MA, Cat# T12921).
After 30 minutes in circulation, mice were re-anesthetized and perfused with 1X phosphate buffer saline (PBS) via the cardiac route. Retinas and livers were isolated and fixed in 4% paraformaldehyde (PFA) for 2 hours prior to immunostaining with FITC-conjugated Lectin.
Biocytin-TMR fluorescence in the livers of corresponding animals were used as internal controls for measurement of Biocytin-TMR fluorescence in the retina as described (Lengfeld et al., 2017; Mazzoni et al., 2017) .
After 30 minutes in circulation, mice were re-anesthetized and perfused with 1X phosphate buffer saline (PBS) via the cardiac route. Retinas and livers were isolated and fixed in 4% paraformaldehyde (PFA) for 2 hours prior to immunostaining with FITC-conjugated Lectin.
Biocytin-TMR fluorescence in the livers of corresponding animals were used as internal controls for measurement of Biocytin-TMR fluorescence in the retina as described (Lengfeld et al., 2017; Mazzoni et al., 2017) .
3
Intracellular Labeling via Electroporation
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Glass pipettes of resistance from 4 to 6 MΩ were filled with a standard pipette solution containing 2%–5% biocytin. These concentrations of biocytin were reached by mixing 4% biocytin (ε-Biotinoyl-L-Lysine; Invitrogen) diluted in some cases with the red dye Alexa 594 (20 µM; Invitrogen) with a solution of 0.8 to 1.5% 5-(and-6)-Tetramethylrhodamine biocytin (Biocytin TMR; Invitrogen).
The tip of the pipette was placed near the selected neuron for electroporation and a loose seal was formed to record extracellular spikes. Spikes were recorded at 5 kHz using a patch-clamp amplifier (npi, Reutlingen, Germany) and Spike2 software (CED, Cambridge, UK). Once a stable configuration was reached, pulses from 300 to 400 mV of 10 ms duration were applied until successful electroporation was verified visually by uptake of the red indicator dye. In addition, we verified in some experiments the viability of the neuron by retesting the responses to visual stimulation after a recovery period of 10–20 min. This recovery period allows sufficient time for the pores formed during the electroporation to reseal, which usually occurs within 1 min [50] (link).
The tip of the pipette was placed near the selected neuron for electroporation and a loose seal was formed to record extracellular spikes. Spikes were recorded at 5 kHz using a patch-clamp amplifier (npi, Reutlingen, Germany) and Spike2 software (CED, Cambridge, UK). Once a stable configuration was reached, pulses from 300 to 400 mV of 10 ms duration were applied until successful electroporation was verified visually by uptake of the red indicator dye. In addition, we verified in some experiments the viability of the neuron by retesting the responses to visual stimulation after a recovery period of 10–20 min. This recovery period allows sufficient time for the pores formed during the electroporation to reseal, which usually occurs within 1 min [50] (link).
4
Evaluating BBB Permeability in Pre-Onset EAE
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The BBB permeability was evaluated in spinal cords, 5 days after transfer of encephalitogenic PLP‐reactive cells, prior to appearance of clinical symptoms (pre‐onset EAE mice, Fig. 1B ). BBB permeability was assessed by 5‐(and 6)‐tetramethylrhodamine biocytin (Biocytin‐TMR, Invitrogen, Thermo Fisher Scientific) extravasation (HICT Pre‐EAE, n = 5; SED Pre‐ EAE, n = 5; naïve controls, n = 3), and adhesion molecules and tight junction molecules expression (HICT Pre‐EAE, n = 5; SED Pre‐ EAE, n = 5). All immunofluorescence analyses were performed by using the Image‐Pro Plus software (Media Cybernetics).
5
In Vivo BBB Permeability Quantification
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To analyze BBB permeability, 1 mg of 5-(and 6)-tetramethylrhodamine biocytin (Biocytin-TMR, T12921, Invitrogen) diluted in 100 μl of PBS was injected per mouse (n = 4 mice/group) intravenously into the tail vein. Seconds after the injection, the ears of injected mice turn pink, indicating the penetration of the material to blood circulation. Thirty minutes after injection, animals were sacrificed, and the brains were extracted and cut as described above. Slides were then stained with CD31 (557355, BD Pharmigen) and DAPI. For each mouse, 8 brain sections were screened (4 brain sections for each Bregma). Images were obtained using Nikon Confocal A1R. Fluorescent-conjugated zymosan A was injected IV to the tail vein followed by mice sacrifice 30 min later.
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