The Journal of Neuroscience, October 25, 2006, ():
DNA Polymerase-
Is Expressed Early in Neurons of Alzheimer's Disease Brain and Is Loaded into DNA Replication Forks in Neurons Challenged with -Amyloid
J. Neurosci. Copani et al.
26: 10949
Supplemental data
Files in this Data Supplement:
- supplemental material
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Fig. S1 - Controls for immunostaining with two mouse monoclonal antibodies. For a full description of immunostaining procedures see material and methods section. The first incubation was done with mouse anti-DNA polymerase β (Neomarkers) detected using PowerVision Poly-HRP-antiMouse/Rabbit/Rat IgG (Immunologic) and developed using DAB (brown). The second incubation was done with either mouse monoclonal 4G8 (Chemicon) or AT8 (Innogenetics), rabbit anti-mouse conjugated with biotin (DAKO) as second step, streptavidin conjugated to alkaline phosphatase (SBA) as third step, and developed using Liquid Permanent Red (DAKO). Between the first and second incubation step, sections were treated with an additional antigen retrieval step (citrate buffer heated by autoclave for 10 min). This step removes the unbound first primary antibodies and inactivates the HRP enzyme activity of the first secondary step. Shown are the following control immunostainings: A) Incubation without the primary antibody (4G8 or AT8) of the second incubation step. In the second incubation, second (rabbit anti-mouse-biotin) and third step (streptavidin-alkaline phosphatase) as well as color development were not omitted. This control shows no reaction between the second step of the second incubation (rabbit anti-mouse conjugated with biotin) and the primary antibody of the first incubation (anti-DNA polymerase β). B) Incubation only without the first primary antibody (anti-DNA polymerase β). Second incubation was done with 4G8 as primary antibody. C) Incubation only without the first primary antibody (anti-DNA polymerase β). Second incubation was done with AT8 as primary antibody. D) Complete double-immunostaining with anti-DNA polymerase β as first primary antibody and 4G8 as second primary antibody. E) Complete double-immunostaining with anti-DNA polymerase β as first primary antibody and AT8 as second primary antibody. F) Incubation without the first antibodies of the first (anti-DNA polymerase β) and second (4G8/AT8) incubation step. All second steps and third step as well as color development were not omitted. G) In this control the sections were incubated without the second step (PowerVision Poly-HRP-antiMouse/Rabbit/Rat IgG) of the first incubation (* condition different from A) and the primary antibody (4G8/AT8) of the second incubation. No reaction between the first primary antibody and the second and third step of the second incubation was observed. This control shows that all unbound first primary antibodies are removed by the citrate and autoclave treatment between the two incubations. All pictures are taken from a case with moderate Alzheimer pathology (Braak 3 for NFT and C for amyloid) and were from the same area in the different sections. Bar represents 60 µm.
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Fig. S2 - Assessment of the homogeneity of the cell population in cultures of rat cortical neurons. For a full description of cell cycle analysis and MAP-2 immunostaining procedures see the methods section. A) Cell cycle distribution profile and MAP-2 immunostaining in a sample culture. B) Cell cycle distribution profile and nonspecific staining by secondary antibody alone in a parallel sample culture. For both (A) and (B) the following plots from the left to the right are shown: 1) FS (forward scatter)/SS (side scatter) dot plot detecting the total PI-stained cell population. The analyzed population is gated in A. 2) Dot plot of confounding cell doublets identified and excluded from the cell cycle analysis. 3) Plot of the DNA profile showing an area of hypoploid DNA corresponding to apoptotic cells and a single peak of G0/G1 cells. 4) Dot plot of FITC-labelled MAP-2 versus PI (A) or FITC-labelled secondary antibody alone versus PI (B). In (B), the nonspecific staining by secondary antibody alone was identified, gated in K, and accounted for 1.9 % of the total cell population. In (A), the nonspecific staining gated in K accounted for 1.36 % of the total cell population; MAP-2+ cells gated in M accounted for 98.64 % of the total cell population.
