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  Indian J Med Microbiol
 

Figure 1: Confocal images of DRG neuron cell bodies' double immunostained for PKC isoforms and TRPV1. PKC isoforms are indicated by the green fluorescence of fluorescein isothiocyanate (FITC) and TRPV1 is indicated by the red fluorescence of Streptavidin Texas Red. Co-localization of PKC isoforms and TRPV1 is shown in overlay with yellow regions. Fluorescence intensity shown as traces represent localization of each PKC isoform (green trace) and TRPV1 (red trace) and is measured across an average of z-series cross-sections of the cell body of the neuron. Cytosolic and plasma membrane distribution of PKCβI and PKCβII are shown. Cytosolic distribution of PKCδ and PKCε are shown. Method: The primary antibodies obtained from Santa Cruz. Their dilutions were rabbit anti-PKC-βI (1/200), rabbit anti-PKC-βII (1/200), and goat anti-VR1 (1/100). Secondary antibodies were goat anti-rabbit-FITC (1/100) (Sigma), rabbit anti-goat biotinylated species (1/100) (Sigma), and Streptavidin Texas Red (1/100) (Amersham Biosciences). All antibodies were diluted in Tris buffered saline (TBS) containing 50 mM Tris and 150 mM NaCl at pH 7.5. Controls included (i) replacement of the primary antibody with normal rabbit serum or (ii) goat serum or (iii) omission of primary antibody. No staining was apparent minus primary antibody or with normal serum controls. All experiments were replicated three times and at least two cultures were examined for each PKC isoform. Cultures were fixed and permeabilized by incubating in cold (4°C) 3.5-4% paraformaldehyde in TBS for 20 min at room temperature (RT), washed three times in TBS, and blocked in blocking buffer 1 [1% bovine serum albumin (BSA), 1% fetal bovine serum (FBS) in TBS) for 1 h at RT. After washing in TBS, cultures to be double immunostained were incubated with the first primary antibody at RT for 1 h, washed in TBS, and incubated with the first secondary antibody (anti-rabbit FITC) for 1 h at RT; it was further incubated with the second primary antibody (goat anti-TRPV1) at 4°C overnight. After washing in TBS, the cultures were incubated in blocking buffer 2 (10% BSA in TBS) for 10 minutes at RT, followed by TBS wash and a two-step incubation with second secondary antibody. The first step involved incubation of the cultures with rabbit anti-goat biotinylated species for 1 h at RT. In the second step, the cultures were washed as previously and incubated with Streptavidin Texas Red for 1 h at RT. After washing, coverslips were inverted on to microscope slides in a 25-μL fluorescence anti-fade solution (Vector Laboratories) and sealed with clear nail polish. Immunostained cultures were visualized by a confocal laser scanning microscope (CLSM) using Leica TCS SP2 System fitted with PL APO 100x/ 1.40-0.7 OIL objective). Sequential scans (FITC and Texas Red) were collected and merged and quantified using Leica TCS SP2 Software ver. 3.0. Fluorescence intensities of FITC and Texas Red were measured across the merged images of neurons as a function of distribution of the specific PKC isoforms and TRPV1, respectively, from the cytosol to the plasma membrane.

Figure 1: Confocal images of DRG neuron cell bodies' double immunostained for PKC isoforms and TRPV1. PKC isoforms are indicated by the green fluorescence of fluorescein isothiocyanate (FITC) and TRPV1 is indicated by the red fluorescence of Streptavidin Texas Red. Co-localization of PKC isoforms and TRPV1 is shown in overlay with yellow regions. Fluorescence intensity shown as traces represent localization of each PKC isoform (green trace) and TRPV1 (red trace) and is measured across an average of z-series cross-sections of the cell body of the neuron. Cytosolic and plasma membrane distribution of PKCβI and PKCβII are shown. Cytosolic distribution of PKCδ and PKCε are shown. Method: The primary antibodies obtained from Santa Cruz. Their dilutions were rabbit anti-PKC-βI (1/200), rabbit anti-PKC-βII (1/200), and goat anti-VR1 (1/100). Secondary antibodies were goat anti-rabbit-FITC (1/100) (Sigma), rabbit anti-goat biotinylated species (1/100) (Sigma), and Streptavidin Texas Red (1/100) (Amersham Biosciences). All antibodies were diluted in Tris buffered saline (TBS) containing 50 mM Tris and 150 mM NaCl at pH 7.5. Controls included (i) replacement of the primary antibody with normal rabbit serum or (ii) goat serum or (iii) omission of primary antibody. No staining was apparent minus primary antibody or with normal serum controls. All experiments were replicated three times and at least two cultures were examined for each PKC isoform. Cultures were fixed and permeabilized by incubating in cold (4°C) 3.5-4% paraformaldehyde in TBS for 20 min at room temperature (RT), washed three times in TBS, and blocked in blocking buffer 1 [1% bovine serum albumin (BSA), 1% fetal bovine serum (FBS) in TBS) for 1 h at RT. After washing in TBS, cultures to be double immunostained were incubated with the first primary antibody at RT for 1 h, washed in TBS, and incubated with the first secondary antibody (anti-rabbit FITC) for 1 h at RT; it was further incubated with the second primary antibody (goat anti-TRPV1) at 4°C overnight. After washing in TBS, the cultures were incubated in blocking buffer 2 (10% BSA in TBS) for 10 minutes at RT, followed by TBS wash and a two-step incubation with second secondary antibody. The first step involved incubation of the cultures with rabbit anti-goat biotinylated species for 1 h at RT. In the second step, the cultures were washed as previously and incubated with Streptavidin Texas Red for 1 h at RT. After washing, coverslips were inverted on to microscope slides in a 25-μL fluorescence anti-fade solution (Vector Laboratories) and sealed with clear nail polish. Immunostained cultures were visualized by a confocal laser scanning microscope (CLSM) using Leica TCS SP2 System fitted with PL APO 100x/ 1.40-0.7 OIL objective). Sequential scans (FITC and Texas Red) were collected and merged and quantified using Leica TCS SP2 Software ver. 3.0. Fluorescence intensities of FITC and Texas Red were measured across the merged images of neurons as a function of distribution of the specific PKC isoforms and TRPV1, respectively, from the cytosol to the plasma membrane.