A comparative antibody analysis of pannexin1 expression in four rat brain regions reveals varying subcellular localizations

Abstract

Pannexin1 (Panx1) channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding patterns. Localizations of Panx1 in brain slices were generated using automated wide field mosaic confocal microscopy for imaging large regions of interest while retaining maximum resolution for examining cell populations and compartments. We compared Panx1 expression over the cerebellum, hippocampus with adjacent cortex, thalamus, and olfactory bulb. While Panx1 localizes to the same neuronal cell types, subcellular localizations differ. Two antibodies with epitopes against the intracellular loop and one against the carboxy terminus preferentially labeled cell bodies, while an antibody raised against an N-terminal peptide highlighted neuronal processes more than cell bodies. These labeling patterns may be a reflection of different cellular and subcellular localizations of full-length and/or modified Panx1 channels where each antibody is highlighting unique or differentially accessible Panx1 populations. However, we cannot rule out that one or more of these antibodies have specificity issues. All data associated with experiments from these four antibodies are presented in a manner that allows them to be compared and our claims thoroughly evaluated, rather than eliminating results that were questionable. Each antibody is given a unique identifier through the NIF Antibody Registry that can be used to track usage of individual antibodies across papers and all image and metadata are made available in the public repository, the Cell Centered Database, for on-line viewing, and download.

Keywords: ATP signaling; connexin; knockout mouse; large field mosaic fluorescent imaging; pannexin channels; paracrine signaling; purinergic receptors.

Figure 1

Diagram of Panx1 topology marked with the epitopes for four different anti-Panx1 antibodies. Anti-Panx1 antibodies recognize the Panx1 protein at different epitopes (Red – Mouse monoclonal 503 anti-Panx1 (Mo503), Blue – Chicken polyclonal Diatheva ANT0027 (CkDia) anti-Panx1, Yellow – Rabbit polyclonal 57 (Rb57) anti-Panx1, Green – Chicken polyclonal 4515 (Ck4515) anti-Panx1). The extracellular loops of Panx1 contain four cysteine residues (magenta) as well as a glycosylation site at N254. A schematized diagram of the carbohydrate tree is indicated at this position. Caspase cleavage sites are located at residues 164–167 (Chekeni et al., ; site A) and 376–379 (Chekeni et al., ; site B) and have been shown to be responsive to Casp3 and Casp7.

Figure 2

Anti-Panx1 and anti-myc antibodies recognize Panx1-myc transiently expressed in HeLa cells. From left to right, each row of images shows anti-Panx1, anti-myc, and a color overlay of the previous two images with Panx1 in green, myc in red, and DAPI in blue, followed by the corresponding DIC image. Note the overlap of the Panx1 and myc staining and the lack of signal in untransfected cells. Each of these antibodies against Panx1 decorates the plasma membrane as well as intracellular localization. A Western blot for Panx1 in cell lysates prepared from over-expressed Panx1 stably expressed in MDCK cells for each of these antibodies is shown in the right hand column.

Figure 3

Western blot analysis of Panx1 protein in cell and brain lysates probed with four different anti-Panx1 antibodies. (A) Banding pattern of MDCK lysates at the far left, glycosylation bands are noted as: GLY0, GLY1, GLY2. (B) Tissue lysates from either brain or spleen from the Genentech generated KO mouse are examined by Western blot with the four antibodies used for imaging studies. The bracket at left highlights the ∼43–55 kDa region where bands for Panx1 species are expected and show decreases in the KO tissue. The Western blot at the far right uses the CT-395 antibody developed by the Laird laboratory and serves as a control for matching against our antibodies. α-tubulin blots are shown as a loading control. (C) Cerebellum and hippocampus tissue lysates from a KOMP generated Panx1 KO show decreased intensity from wild type in Western blots. Here, Western blotting against GAPDH was used as a loading control.

Figure 4

Large scale mosaic imaging of rat brain cerebellum. Top: this representative cerebellum montage is labeled with CkDia anti-Panx1 antibody (green), GFAP (red), and DAPI (blue, nuclei). This mosaic image is made up of 587 tiles. Each tile is a maximum intensity projection of a stack of five Z-sections that were stitched together to reconstruct this single, high-resolution 2D image. Bottom: full resolution views of cerebellar regions and cell types labeled by the four anti-Panx1 antibodies. Arrowheads in the Mo503 left hand image = stellate cells.

Figure 5

Cerebellar cell types labeled by Rb57 anti-Panx1 antibody. Labeling of rat cerebellum tissue with the Rb57 anti-Panx1 antibody reveals expression in Purkinje neurons (white arrowheads), Golgi cells within the granular layer (white arrows), Bergmann glia within the molecular layer of the cerebellum (yellow arrowheads), and additional cell bodies in the molecular layer that may be stellate cells. (A) Merged image (B) Panx1 channel shown in grayscale.

Figure 6

Large scale mosaic imaging of hippocampus and neocortex lying adjacent to the hipps ocampus. Top: this Representative hippocampus montage is labeled with CkDia anti-Panx1 antibody (green), GFAP (red), and counterstained nuclei with DAPI (blue). This mosaic image is made up of 167 tiles. Each tile is a maximum intensity projection of a stack of four Z-sections that were stitched together to reconstruct this single, high-resolution 2D image. Bottom: full resolution views of hippocampal regions and cell types labeled by the four anti-Panx1 antibodies.

Figure 7

Differential subcellular labeling of Panx1 in the dendate gyrus of the rat hippocampus with combinations of four anti-Panx1 antibodies. Although the neurons of the hippocampus are a consensus cell type labeled by all four of the Panx1 antibodies, there are differences in the apparent subcellular localization of the protein population recognized by each antibody. In these five panels, each antibody is shown in black and white (middle and right hand images) and as a composite color image in the left hand images. In all composite images, astrocytes are labeled with anti-GFAP (blue). (A) Co-labeling with Ck4515 and Mo503. (B) Co-labeling with Ck4515 and Rb57. (C) Co-labeling with Rb57 and Mo503. (D) Co-labeling with CkDia and Mo503. (E) Co-labeling with Rb57 and CkDia. Tissue slices labeled with Rb57 (B,C,E) underwent antigen retrieval prior to immunolabeling. Co-labeling tissue with the polyclonal antibodies reveals that these primarily highlight the cell body and the adjacent portion of the dendrite however the Mo503 antibody (red) decorates the long dendrites of these cells with less noticeable staining at the cell bodies. However, there is a substantial degree of overlap in staining between all the antibodies.

Figure 8

Large scale mosaic imaging of rat brain olfactory bulb and neocortex lying adjacent to the olfactory bulb. Top: representative montage labeled with CkDia anti-Panx1 antibody (green), anti-GFAP (red), and DAPI (blue, nuclei). This mosaic image is made up of 649 tiles. Each tile is a maximum intensity projection of a stack of five Z-sections that were stitched together to reconstruct this single, high-resolution 2D image. Bottom: full resolution views of olfactory bulb regions and cell types labeled by the four anti-Panx1 Abs. White arrows = red blood cells, MOB, main olfactory bulb; AOB, accessory olfactory bulb; NC, neocortex.

Figure 9

Large scale mosaic imaging of rat brain thalamus. Top: representative thalamus montage labeled with CkDia anti-Panx1 (green), anti-GFAP (red), and DAPI (blue, nucleus). This mosaic image is made up of 447 tiles. Each tile is a maximum intensity projection of a stack of five Z-sections that were stitched together to reconstruct this single, high-resolution 2D image. Bottom: full resolution views of thalamus regions labeled by the four anti-Panx1 antibodies.