Seven-micrometer-thick paraffin-embedded cross sections of human organotypic skin cultures were deparaffinized and rehydrated by two changes of xylene and washed with descending ethanol concentrations. For antigen retrieval, the cross sections were boiled twice in demasking buffer (10 mM sodium citrate, pH 6.0, with 0.05% Tween-20) followed by blocking in PBS with 10% BCS for 20 min at RT. Antibodies were diluted in PBS with 1% BCS and incubated with the sections for 1 h. Primary antibodies included the following: filaggrin (sc-66192; Santa Cruz) at 1:50 dilution, MTA2 (ab8106; Abcam) at 1:500 dilution, and keratin 10 (MS-611-P; NeoMarkers) at 1:400 dilution. Alexa Fluor 555–conjugated goat anti-mouse (A21422, 1:300; Life Technologies) was used as a secondary antibody diluted in PBS with 1% BCS. Unbound antibodies were washed off after 1 h of incubation with PBS (three times, 5 min, RT). Slides were mounted in DAPI Fluoromount-G (Biozol). Finally, cross sections were analyzed with a BZ-X800 fluorescence microscope and BZ-X800 Analyzer software (Keyence).
Dapi fluoromount g
Manufactured by Calibre Scientific
DAPI Fluoromount-G is a mounting medium designed for fluorescence microscopy applications. It contains the DNA-binding dye DAPI, which fluoresces blue when bound to DNA, allowing for the visualization of nuclei in fixed samples.
Automatically generated - may contain errors
Lab products found in correlation
Bz x800 fluorescence microscope, by Keyence (1 mentions)
Filaggrin sc 66192, by Santa Cruz Biotechnology (1 mentions)
Bz x800 analyzer software, by Keyence (1 mentions)
Alexa 568, by Thermo Fisher Scientific (1 mentions)
Anti mouse alexa 488, by Thermo Fisher Scientific (1 mentions)
Dp50 digital camera, by Olympus (1 mentions)
Mab377, by Thermo Fisher Scientific (1 mentions)
A21202, by Thermo Fisher Scientific (1 mentions)
Ab2904, by Abcam (1 mentions)
Axiovert m200, by Zeiss (1 mentions)
Ab1316, by Abcam (1 mentions)
Vt1200, by Leica (1 mentions)
Ab145311, by Abcam (1 mentions)
4 protocols using dapi fluoromount g
1
Immunohistochemistry analysis of human skin
2
Quantifying TRPV1+ Neurons in DRG
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For the therapeutic setting, a minimum of six and a maximum of eight DRGs per segment (lumbar, thoracic and cervical) were dissected and washed with PBS, stored in 4% paraformaldehyde (PFA) for 1 h and cryoprotected in 30% sucrose overnight. Cryosections (8 µm) were stained with primary antibodies against TRPV1 (1:500, Thermo Fisher Scientific, PA1-29421) and NeuN (1:300, Merck Millipore, MAB377), anti-rabbit Alexa 568 (1:1000, Invitrogen) to visualize TRPV1 staining and anti-mouse Alexa 488 (1:1000, Invitrogen) as a secondary antibody for NeuN. DAPI Fluoromount-G (Biozol) was used to visualize cell nuclei. The fluorescence signal of TRPV1 was detected using an inverted fluorescence microscope (BX51; Olympus) equipped with an Olympus DP50 digital camera. Three replicate pictures of each segment/rat were recorded three times (biological n = 4, technical n = 9, ×20 magnification) and analysed by two blinded investigators using the image analysis software ImageJ (National Institutes of Health, Bethesda, USA). TRPV1high+ cells were counted and reported as the percentage of all neuronal cells in DRG.
3
Immunofluorescence Imaging of Aortic Angiostatin and VEGF
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Paraffin sections (3 µm) of aortic vessels from WT and α-GAL-Tg/KO mice were deparaffinized and incubated in a 10 mM citrate buffer at 95°C for 40 min for antigen retrieval. Afterward, the sections were incubated for 10 min in 0.5% Triton/TBS for permeabilization, washed in TBS, and quenched in a 0.25% Sudan black solution for 30 min on a shaker in the dark. After repeated washing, the sections were blocked in 3% bovine serum albumin (BSA) in TBS for 1 h at room temperature. The sections were then incubated with primary antibodies [anti-angiostatin (Abcam ab2904), 1:20 in 3% BSA or anti-VEGFα (Abcam, ab1316), 1:400 in 3% BSA] overnight at 4°C, washed in TBS, and incubated with secondary antibodies [donkey anti-mouse Alexa Fluor 488 (A21202 life tech) or donkey anti-rabbit Alexa Fluor 488 (21206 life tech), 1:500 in 3% BSA] for 2 h at room temperature. After repeated washing in TBS, the slides were mounted with DAPI Fluoromount G (Biozol) and fluorescence signals were descriptively analyzed using a Zeiss Axiovert M200 with ApoTome.
4
Histological Reconstruction of Electrode Tracks
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For histological reconstruction of the electrode track, the probe was removed and re-inserted in the same location coated with DiI (Abcam-ab145311) diluted in ethanol. The animal was then sacrificed either with isoflurane (>4%) or a subcutaneous injection of a Ketamine-Xylazine mix (Ketamidor 1 g/mL, Rompun 2%). Cardiac perfusion was performed with phosphate buffer saline solution (PBS) followed by 4% paraformaldehyde (PFA) in PBS. The brains were post-fixed overnight in 4% PFA and stored in PBS until histological slicing was performed using a vibratome (Leica VT1200 S). The brain slices were mounted in DAPI-Fluoromount-G (70–100 µm slices, Biozol Cat. 0100-20). Perfused zebra finch brains were transferred to 15% sucrose in PBS for 24 h post-fixation, and then they were moved into 30% sucrose for at least 12 h prior to sectioning with a cryostat microtome. The optic tectum was sliced into 90 µm sagittal sections, mounted using DAKO (Agilent), and the recording location was confirmed by visually inspecting the recording track post hoc in brain slices.
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