Journal: bioRxiv

Article Title: Single-cell transcriptional landscape of temporal neutrophil response to burn wound in larval zebrafish

doi: 10.1101/2024.04.01.587641

Figure Lengend Snippet: (A) Schematic view of validating signatures identified from zebrafish myeloid subsets in human burn patient expression profile. (B) Expression level distribution of Immature_Neuts signatures (top panel) and lgals3bpb_Mac1 (bottom panel) by patient TBSA shown as raincloud plots. Paired T-tests were performed between the Low and the Very High TBSA groups. (C) Expression distribution of GPR84 and GYG1 in patients by TBSA level. Spearman correlation (ρ) is calculated between gene expression and TBSA level. (D) Representative flow plots (left) and quantifications of frequencies shown as percentages (right) of GPR84+ neutrophils in burn (n = 6) and healthy (n = 4) blood. Paired samples t-test, p = 0.022.

Article Snippet: Protein-targeted staining was done with the following antibodies in Cell Staining Buffer: Alexa Fluor 700-conjugated anti-human CD16 at 1:400, PE/Fire-640-conjugated anti-human CD66b at 1:400, PerCP-conjugated anti-human CD3 at 1:200, PerCP-conjugated anti-human CD19 at 1:200, PerCP-conjugated anti-human CD56 at 1:400, PerCP-conjugated anti-human CD203c at 1:100 (BioLegend), BUV395-conjugated mouse anti-human CD45 at 1:100 (BD Biosciences), and PE-conjugated GPR84 at 1:200 (Alomone Labs).

Techniques: Expressing, Gene Expression

Journal:

Article Title: Protein tyrosine kinase is expressed and regulates ROMK1 location in the cortical collecting duct

doi: 10.1152/ajprenal.00301.2003

Figure Lengend Snippet: Confocal images showing the colocalization of aquaporin-2 (AQP2; green) and c-Src (red) in the renal cortex (A) and OM (B) from rats on a normal-K diet. Bar = 10 µM or as indicated.

Article Snippet: Immunocytochemical staining The slides were washed with 1× PBS for 15 min and permeablized with 0.4% Triton dissolved in 1× PBS buffer containing 1% BSA and 0.1% lysine (pH 7.4) for 15 min. Kidney slices were blocked with 2% goat serum for 1 h at room temperature and then incubated with ROMK antibody (Alomone, Jerusalem, Israel), c-Src monoclonal antibody (Transduction Lab), aquaporin-2 (AQP2) antibody (Alomone), or Tamm-Horsfall glycoprotein (THP) antibody (ICN, Pharmaceutical, Aurora, OH) for 12 h at 4°C.

Techniques:

Journal:

Article Title: Protein tyrosine kinase is expressed and regulates ROMK1 location in the cortical collecting duct

doi: 10.1152/ajprenal.00301.2003

Figure Lengend Snippet: Magnified confocal image (×100) showing the double staining of AQP2 and c-Src in the renal cortex (A) and OM (B). Bar = 10 µM.

Article Snippet: Immunocytochemical staining The slides were washed with 1× PBS for 15 min and permeablized with 0.4% Triton dissolved in 1× PBS buffer containing 1% BSA and 0.1% lysine (pH 7.4) for 15 min. Kidney slices were blocked with 2% goat serum for 1 h at room temperature and then incubated with ROMK antibody (Alomone, Jerusalem, Israel), c-Src monoclonal antibody (Transduction Lab), aquaporin-2 (AQP2) antibody (Alomone), or Tamm-Horsfall glycoprotein (THP) antibody (ICN, Pharmaceutical, Aurora, OH) for 12 h at 4°C.

Techniques: Double Staining

Journal:

Article Title: Immunolocalization of the Voltage-gated Potassium Channel Kv2.2 in GABAergic Neurons in the Basal Forebrain of Rats and Mice

doi: 10.1002/cne.22457

Figure Lengend Snippet: Expression of Kv2.2 in the neurons of the magnocellular preoptic nucleus (MCPO) and the horizontal band of Broca (HDB). A–C: Expression of Kv2.2 in the NeuN-positive neuronal population in the basal forebrain. Scale bar, 50 μm. D–E: Nickel enhanced 3-3′diaminobenzidine (DAB) immunostaining was used to determine specific localization of Kv2.2-expressing neurons. Rat coronal sections (8.9 and 9.2mm anterior to the interaural line) were immunostained with the anti-Kv2.2 antibody. Anatomical landmarks such as the anterior commissure (indicated by asterisk) were used to locate the expression of Kv2.2 in the MCPO/HDB nuclei.

Article Snippet: For DAB staining, sections were incubated with rabbit anti-Kv2.2 antibody (0.3 μg/ml, Alomone Labs, Jerusalem, Israel) overnight, washed, and probed with a biotinylated anti-rabbit secondary antibody (1:250, Vector Labs, Burlingame, CA).

Techniques: Expressing, Immunostaining

Journal:

Article Title: Immunolocalization of the Voltage-gated Potassium Channel Kv2.2 in GABAergic Neurons in the Basal Forebrain of Rats and Mice

doi: 10.1002/cne.22457

Figure Lengend Snippet: Enriched expression of Kv2.2 in non-cholinergic neurons in the MCPO/HDB of the mouse brain. A: Nickel enhanced DAB immunostaining of Kv2.2-immunoreactive neurons in the mouse brain. A coronal section (3.94 mm anterior to the interaural line) was immunostained with the anti-Kv2.2 antibody. B: Corresponding mouse brain atlas to A. VLPO, ventrolateral preoptic nucleus; LPO, lateral preoptic nucleus; VP, ventral pallidum; AC, anterior commissure; SIB, substantia innominata basal; Tu, olfactory tubercle. C–E: Confirmation of the enrichment of Kv2.2 in the MCPO in immunofluorescence staining. Mouse coronal sections were double immunostained with K89 anti-Kv2.1 and anti-Kv2.2 antibodies. F–H: Reciprocal expression of Kv2.2 and ChAT in the MCPO of mouse. Mouse coronal sections were double immunostained with anti-Kv2.2 and anti-ChAT antibodies. Scale bars, 100 μm.

Article Snippet: For DAB staining, sections were incubated with rabbit anti-Kv2.2 antibody (0.3 μg/ml, Alomone Labs, Jerusalem, Israel) overnight, washed, and probed with a biotinylated anti-rabbit secondary antibody (1:250, Vector Labs, Burlingame, CA).

Techniques: Expressing, Immunostaining, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Increased Migration of Monocytes in Essential Hypertension Is Associated with Increased Transient Receptor Potential Channel Canonical Type 3 Channels

doi: 10.1371/journal.pone.0032628

Figure Lengend Snippet: A ; Immunoblot showing specificity of antibodies against TRPC3 in monocytes from normotensive control subjects (NT) and patients with essential hypertension (HT) in the absence or presence of TRPC3 antigens (TRPC3+Ag). The predicted molecular weight of TRPC3 is 97 kDa. B ; Immunoblot showing specificity of antibodies against TRPC3 in monocytes from normotensive control subjects (NT, n = 8), patients with type 2 diabetes mellitus (DM, n = 9), patients with essential hypertension (HT, n = 8) or hypertensive patients with type 2 diabetes mellitus (HT+DM, n = 10). Summary data of the TRPC3 expression (normalized to GAPDH). *p<0.05, compared to NT. Data are mean ± SEM. C ; Representative in-cell western assay and summary data of the TRPC3 expression (normalized to CD14 expression used as an internal reference) in monocytes from normotensive control subjects (Normotensive, and opened bars, n = 3) and patients with essential hypertension (Hypertensive, filled bars, n = 3) under control conditions and after transfection with scrambled siRNA or specific siRNA against TRPC3 for 48 h. In-cell western assay was performed using specific antibodies and fluorescence-labeled secondary antibodies. TRPC3 (visible in green) normalized to CD14 (used as an internal reference). Measurements were performed in duplicate for each sample. *p<0.05 or **p<0.01 for the comparison with their controls; and ## p<0.01 for the comparison Hypertensive (filled bars) vs. Normotensive (open bars). D ; Representative in-cell western assay and summary data of the TRPC3 and TRPC6 expression in monocytes from normotensive control subjects under control conditions and after transfection with specific siRNA against TRPC3 for 48 h. In-cell western assay was performed using specific antibodies and fluorescence-labeled secondary antibodies. TRPC3 and TRPC6 expression (visible in green) normalized to CD14 (visible in red used as an internal reference). Measurements were performed in duplicate for each sample. **p<0.01 compared to control conditions. Data are mean ± SEM of three independent experiments. E ; Summary data of the fMLP-induced monocyte migration from hypertensive patients (HT, filled bars) and normotensive control subjects (NT, opened bars) quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. Monocytes chemotaxis was expressed as the mean number of migrated cells per high-power fields from duplicate wells. Experiments were performed under control conditions, after transfection with scrambled siRNA or specific siRNA against TRPC3. *p<0.05; **p<0.01 compared to normotensive control subjects under control conditions. Data are mean ± SEM of eight independent experiments. F ; Spontaneous migrations of monocytes from normotensive control subjects (NT; open bars) and hypertensive patients (HT, filled bars) were tested using medium or after transfection with scrambled siRNA or specific siRNA against TRPC3. The data was quantified by counting the number of cells that had completely migrated through the membrane in six random high-power fields (HPF, 40×) per well. P>0.05 compared to NT. Data are percent of medium as mean ± SEM of three independent experiments.

Article Snippet: After that incubated with rabbit anti-TRPC3 antibodies (1∶1000, Alomone Laboratories, Jerusalem, Israel) as the primary antibodies for 2 h, washed, incubated with IRDye 800 CW infrared fluorescent dye conjugated goat anti-rabbit antibodies (1∶1000, Biomol, Hamburg, Germany) as the secondary antibody overnight and washed, and quantitative imaging was performed at 810 nm emission with an excitation wavelength of 780 nm.

Techniques: Western Blot, Molecular Weight, Expressing, In-Cell ELISA, Transfection, Fluorescence, Labeling, Migration, Chemotaxis Assay

Journal: PLoS ONE

Article Title: Increased Migration of Monocytes in Essential Hypertension Is Associated with Increased Transient Receptor Potential Channel Canonical Type 3 Channels

doi: 10.1371/journal.pone.0032628

Figure Lengend Snippet: A , B ; fMLP activates ERK or phosphorylation of ERK ( A ) and Akt or phosphorylation of Akt ( B ) in a dose- and time-dependent manner in monocytes from normotensive control subjects. 10 nmol/L open bars, 100 nmol/L filled bars. Data are mean ± SEM, n = 3. *p<0.05 compared to lower concentration conditions. C , D ; Increased fMLP-induced phosphorylation of ERK ( C ) and Akt ( D ) in monocytes from patients with essential hypertension. The proteins were measured using immunoblotting with specific antibodies. Data are mean ± SEM from three independent experiments. *p<0.05 compared to normotensive control subjects. E ; fMLP activates monocytes by an ERK-dependent and Akt-dependent pathway. Akt, ERK, or pERK and pAkt were measured using immunoblotting with specific antibodies. In the presence of 2-APB or after administration of specific siRNA against TRPC3, the fMLP-induced ERK, pERK; Akt and pAkt were significantly reduced when compared with control conditions. Data are mean ± SEM from six independent experiments. *p<0.05; **p<0.01 compared to control.

Article Snippet: After that incubated with rabbit anti-TRPC3 antibodies (1∶1000, Alomone Laboratories, Jerusalem, Israel) as the primary antibodies for 2 h, washed, incubated with IRDye 800 CW infrared fluorescent dye conjugated goat anti-rabbit antibodies (1∶1000, Biomol, Hamburg, Germany) as the secondary antibody overnight and washed, and quantitative imaging was performed at 810 nm emission with an excitation wavelength of 780 nm.

Techniques: Concentration Assay, Western Blot