Anti-Parvalbumin antibody (ab11427)

Cell type specific markers do not reveal abnormalities in retinal cell number or localization in shNr1d1 injected mice

Labeling with markers for Blue opsin (blue (S-) opsin expressing cones), Green opsin (green (M-) opsin expressing cones), Rhodopsin (Rod cells), Parvalbumin (amacrine cells), Calbindin (horizontal cells), and Brn3a (ganglion cells) did not reveal any problems with cell placement, organization or morphology within the retina when examined at postnatal day 30.5. n = 3. Scale bar  = 50 µm.

Parvalbumin is detected using ab114427 at 1/200 dilution following fixation with methanol:acetic acid (1:3) and embeding with paraffin.

Immunocytochemistry/Immunofluorescence analysis of U251 cells labeling Parvalbumin (green) with ab11427 at 1/200. F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue). Cells were fixed with formaldehyde and incubated with the primary antibody overnight at 4°C. A DyLight 488-conjugated secondary antibody was used. 60X magnification. Right - negative control.

Passive clarity technique (PACT) immunostaining and 3D imaging of mouse brain slices at differernt temperatures using ab11427 and nuclei were counterstained with DAPI (blue). Tissues were then incubated in sorbitol for 5 hours and mounted for imaging. Scale bar, 100 µm.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labeling Parvalbumin (green) with ab11427 at 1/100. F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue). Cells were fixed with formaldehyde and incubated with the primary antibody overnight at 4°C. A DyLight 488-conjugated secondary antibody was used. 60X magnification. Right - negative control.

Immunocytochemistry/Immunofluorescence analysis of C6 cells labeling Parvalbumin (green) with ab11427 at 1/100. F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue). Cells were fixed with formaldehyde and incubated with the primary antibody overnight at 4°C. A DyLight 488-conjugated secondary antibody was used. 60X magnification. Right - negative control.

Immunocytochemcial immunofluorescence analysis of 4% PFA & 0.2% Picric acid fixed rat cordical cells in culture, labelling parvalbumin with ab11427 at a dilution of 1/500 incubated for 12 hours at 4°C in 10mM PBS & 0.03% Triton X diluent blend. The secondary was a Donkey anti-Rabbit polyclonal Alexa Fluor^® 488 conjugate at 1/200.

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ab11427 staining mouse brain cells by ICC/IF.  Cells were PFA fixed and permeabilized with Triton, prior to blocking with 10% serum for 1 hour at RT.  The primary antibody was used undiluted and incubated with the sample for 18 hours at 4°C.  An Alexa Fluor® 488 conjugated goat anti-rabbit antibody was used as the secondary.

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3D volumetric imaging of mouse brain slices using 3 different hydrogel formulas (A4B4P4, A1B1P4 and A4B4P0) to show development of enhanced CLARITY methods for peripheral tissues.
The A1B1P4 formulation improves the penetration rate of antibodies, resulting in full 1 mm brain slices stained with 12hrs incubation in ab11427, visualized using the marker Parvalbumin (PV). Scale bar = 100 μm.

Staining quality was quantified by measuring average cellular signal intensity normalized by average extracellular background intensity. 6 samples per gel type.

Immunohistochemistry was performed on normal biopsies of deparaffinized Human cerebellum tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:100 with a rabbit polyclonal antibody recognizing Parvalbumin ab114227 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.