Journal: International Archives of Allergy and Immunology

Article Title: Expression of T-Type Voltage-Gated Calcium Channel in the Cilia of Human Nasal Epithelial Cells

doi: 10.1159/000521765

Figure Lengend Snippet: Photomicrographs of fluorescence immunohistochemical staining. The nasal turbinate specimens were fixed with 4% paraformaldehyde and embedded while frozen in Tissue-Tek OCT Compound. Seven-micrometer-thick sections were prepared and mounted on silane-coated glass slides. The sections were incubated with rabbit anti-human Cav3.1 (CACNA1G) polyclonal antibody, rabbit anti-human Cav3.2 (CACNA1H) polyclonal antibody, rabbit anti-human Cav3.3 (CACNA1I) polyclonal antibody, or rabbit anti-human Cav2.3 (CACNA1E) polyclonal antibody. As a negative control, the primary antibodies were omitted from the process. The sections were then reacted with a secondary antibody, Alexa Fluor 488-conjugated goat anti-rabbit IgG, coverslipped with Prolong Gold antifade reagent containing DAPI, and examined under a Carl Zeiss Axioskop 2 Plus fluorescence microscope. Images were captured using a Carl Zeiss AxioCam digital camera attached to the microscope. Green and blue colors express the fluorescence of Alexa Fluor 488 and DAPI, respectively. Moderate fluorescence for Cav3.1 and Cav3.3 is observed on the surface of the epithelial cells and nasal gland cells ( a, b ), whereas there is no fluorescence for Cav3.2 or Cav2.3 ( c, d ). Scale bar, 20 μm.

Article Snippet: The sections were then treated with 1% normal BSA in PBST for 1 h and incubated with rabbit anti-human Cav3.1 (CACNA1G) polyclonal antibody (Cusabio) or rabbit anti-human Cav3.3 (CACNA1I) polyclonal antibody (Novus Biologicals) at 4°C overnight.

Techniques: Fluorescence, Immunohistochemical staining, Staining, Incubation, Negative Control, Microscopy

Journal: International Archives of Allergy and Immunology

Article Title: Expression of T-Type Voltage-Gated Calcium Channel in the Cilia of Human Nasal Epithelial Cells

doi: 10.1159/000521765

Figure Lengend Snippet: Immunoelectron microscopy. The nasal turbinate specimens were fixed with 4% paraformaldehyde and embedded while frozen in Tissue-Tek OCT Compound. Seven-micrometer-thick sections were prepared using a cryostat and mounted on silane-coated glass slides. The sections were incubated with rabbit anti-human Cav3.1 (CACNA1G) polyclonal antibody or rabbit anti-human Cav3.3 (CACNA1I) polyclonal antibody, fixed with 1% glutaraldehyde, and developed with DAB. As a negative control, the primary antibodies were omitted from the process. The sections were then post-fixed with 1% OsO 4 and embedded in Epon 812. Seventy- to 100-nm-thick ultrathin sections were prepared using an ultramicrotome, stained with 4% lead nitrate, and examined under a JEOL JEM-1200EX transmission electron microscope. a, b Photomicrographs of DAB immunohistochemical staining. Immunoreactivities for both Cav3.1 ( a ) and Cav3.3 ( b ) are observed on the surface of the epithelial cells. Scale bar, 50 μm. c Immunoelectron micrographs for Cav3.1 and Cav3.3. Immunoreactivity for Cav3.1 was localized on the surface of the cilia, and that for Cav3.3 was localized in the cilia and at the base of the cilia. Scale bar, 0.5 μm.

Article Snippet: The sections were then treated with 1% normal BSA in PBST for 1 h and incubated with rabbit anti-human Cav3.1 (CACNA1G) polyclonal antibody (Cusabio) or rabbit anti-human Cav3.3 (CACNA1I) polyclonal antibody (Novus Biologicals) at 4°C overnight.

Techniques: Immuno-Electron Microscopy, Incubation, Negative Control, Staining, Transmission Assay, Microscopy, Immunohistochemical staining

Journal: International Archives of Allergy and Immunology

Article Title: Expression of T-Type Voltage-Gated Calcium Channel in the Cilia of Human Nasal Epithelial Cells

doi: 10.1159/000521765

Figure Lengend Snippet: Western blot analysis. The collected turbinate mucosa was minced/ground and soaked in the RIPA lysis buffer with protease inhibitor and phosphatase inhibitor. The lysate was centrifuged, and the supernatant was mixed with EzApply and boiled. An aliquot of the mixture equivalent to 10 μg protein was loaded onto the well of a 5–15% gradient polyacrylamide gel and electrophoresed at a constant current of 20 mA/gel for 80 min. The gel was then electrotransferred onto a nitrocellulose membrane at a constant current of 180 mA/gel for 60 min. The blotted membrane was incubated with rabbit anti-human Cav3.1 (CACNA1G) polyclonal antibody, rabbit anti-human Cav3.3 (CACNA1I) polyclonal antibody, or mouse anti-human β-actin monoclonal antibody. The membrane was reacted with peroxidase-conjugated goat anti-rabbit or anti-mouse IgG and soaked in Luminescence solutions for chemiluminescence detection. Images were captured by a LumiCube CMOS digital camera.

Article Snippet: The sections were then treated with 1% normal BSA in PBST for 1 h and incubated with rabbit anti-human Cav3.1 (CACNA1G) polyclonal antibody (Cusabio) or rabbit anti-human Cav3.3 (CACNA1I) polyclonal antibody (Novus Biologicals) at 4°C overnight.

Techniques: Western Blot, Lysis, Protease Inhibitor, Membrane, Incubation

Journal: International Archives of Allergy and Immunology

Article Title: Expression of T-Type Voltage-Gated Calcium Channel in the Cilia of Human Nasal Epithelial Cells

doi: 10.1159/000521765

Figure Lengend Snippet: Expressions of CACNA1G mRNA and CACNA1I mRNA. The collected turbinate mucosa was minced and soaked in TRIzol reagent. Total RNA was extracted by the acid guanidiniumthiocyanate-phenol-chloroform method, cleaned up with a BioRobot EZ1 system, and reverse-transcribed to cDNA using a High-Capacity RNA-to-cDNA Kit. The real-time reverse transcription-polymerase chain reaction analysis was performed with an Applied Biosystems StepOnePlus real-time PCR system using TaqMan Fast Universal PCR Master Mix with glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) as a housekeeping gene. The thermal cycler conditions were as follows: holding at 95°C for 2 min, followed by two-step polymerase chain reaction of 40 cycles of 95°C for 1 s and 60°C for 20 s. The measured threshold cycle (C T ) was normalized by subtracting the C T for GAPDH of each sample from those for CACNA1G and CACNA1I . From the obtained ΔC T , the ratio of the target mRNA to GAPDH mRNA was calculated by the following formula: target mRNA/ GAPDH mRNA ratio = 2 −ΔC T . a Representative trace of amplification plots of real-time RT-PCR. b Expression levels of CACNA1G mRNA and CACNA1I mRNA. The average ratios of CACNA1G mRNA/GAPDH mRNA and CACNA1I mRNA/GAPDH mRNA are 0.0088 ± 0.0075 and 0.0110 ± 0.0072, respectively ( n = 8).

Article Snippet: The sections were then treated with 1% normal BSA in PBST for 1 h and incubated with rabbit anti-human Cav3.1 (CACNA1G) polyclonal antibody (Cusabio) or rabbit anti-human Cav3.3 (CACNA1I) polyclonal antibody (Novus Biologicals) at 4°C overnight.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Expressing