Figure 1.
Raising tau isoform-specific antibodies.
(A) Schematic representation of the exon structure of the MAPT locus that encodes murine tau. Alternative splicing of exons 2, 3, and 10 generates the three isoforms 0N4R, 1N4R, and 2N4R that are present in the adult murine brain. The scheme shows the location of the epitopes that were used to raise specific antibodies for 0N, 1N and 2N murine tau, as well as for total murine tau (M), without cross-reactivity with human tau. (B) Western blot analysis of RAB-soluble tau extracts obtained from brains of 2-months old wild-type (WT) mice, with stripes probed separately with Tau5, M (total mouse tau) and the murine tau isoform-specific antibodies 01, 2N, and 2N reveals their specificity. Tau knock-out (KO) tissue was included as negative control.
Figure 2.
Western blot analysis of dephosphorylated extracts obtained from dissected tissues of 2-month old mice using the Tau5 antibody.
(A) Analysis of wild-type (WT) mice; brain (bra), pancreas (pan), liver (liv), kidney (kid), muscle (mus), spleen (spl), testis (tes), and heart (hea). (B) Inclusion of tau knock-out (KO) tissue. Molecular weight and isoforms are indicated. (C) Western blot analysis of dephosphorylated brain extracts from 2-month old, 2-week old and P0 WT mice using the 3R- and 4R-specific antibodies RD3 and RD4, respectively. Note: The relatively intensities of RD3 and RD4 cannot be used to deduce the relative levels of the 3R and 4R isoforms. Instead, the Tau5 pattern is informative.
Figure 3.
Western blot analysis of dephosphorylated samples derived from different brain areas of 2-month old WT mice using the Tau5 antibody.
(A) Brain tissues: cortex (ctx), hippocampus (hip), pituitary gland (pit), striatum (str), cerebellum (cer) and olfactory bulb (olf). (B) Relative levels of tau isoforms in the different brain areas. (C) Significance analysis using two-way ANOVA. The significance level is calculated by comparing to cortex. *, P< 0.05, **, P< 0.01, ***, P< 0.001, and ns, not significant. Error bars represent the standard error of the mean (SEM).
Figure 4.
Subcellular fractionation of brains from 2-month old WT mice.
(A) Relative purity of the cytoplasmic (C), membrane (M), nuclear (N), chromatin-bound (nuclear) (Ch) and cytoskeletal (S) fractions were confirmed using the following antibodies: GAPDH (for C), GLT-1 (for M), hnRNP-1 (for N), histone H2A (for Ch), and GFAP (for S). (B) Western blot analysis of dephosphorylated brain fractions obtained from 2-month old WT mice using the tau-specific Tau5 antibody. (C) Relative ratio of the three tau isoforms in the five fractions. (D) Significance analysis using two-way ANOVA. The significance level is calculated by comparing to the cytoplasmic fraction. *, P< 0.05, **, P< 0.01, ***, P< 0.001, and ns, not significant. Error bars represent the standard error of the mean (SEM).
Figure 5.
Subcellular fractionation of brains from 2-week old WT mice.
(A) Western blot analysis using the same subcellular markers as used in Figure 4. (B) Western blot analysis of dephosphorylated brain fractions obtained from 2-week old WT mice using Tau5. Note: At 2 weeks, there is also expression of the fetal (3R) isoform. The 0N3R and 0N4R isoforms have been assessed 'collectively'. (C) Relative ratio of the three tau isoforms in the five fractions. (D) Significance analysis using two-way ANOVA.
Figure 6.
Subcellular fractionation of brains from WT mice at P0.
At this stage, 4R isoforms are not expressed. 0N3R is the predominant species, and 2N3R is not detected. (A) Western blot analysis using the same subcellular markers as in Figure 4. (B) Western blot analysis of dephosphorylated brain fractions obtained from P0 WT mice using Tau5. (C) Relative ratio of the three tau isoforms in the fractions. (D) Significance analysis using two-way ANOVA.
Figure 7.
Relative ratio of tau isoforms in five subcellular fractions at P0, 2 weeks and 2 months of age.
(A) Cytoplasmic, (B) membrane, (C) soluble nuclear, (D) chromatin-bound, and (E) cytoskeletal fraction. Error bars represent the standard error of the mean (SEM).
Figure 8.
Immunohistochemical analysis of tau isoforms in 2 month-old wild-type mice.
(A-D) Testing of antibodies reveals no bleach-through. Staining of the hippocampus with (A) Tau5 and (C) Dako tau. Omitting the primary antibodies and reacting the sections only with the secondary antibodies (B) Alexa Fluor 555 goat anti-mouse IgG, or (D) Alexa Fluor 488 goat anti-rabbit IgG. (E-H) Pre-absorption with the peptides, with which the corresponding antibodies pan-tau M (E), 0N (F), 1N (G) and 2N (H) were generated. (I-L) Staining with the new antibodies M (I), 0N (J), 1N (K) and 2N (L) in red, (M-P) counter-staining with Dako tau in green, (Q-T) Merged images. (U-X) Sections from tau knock-out mice used as negative control for antibodies pan-tau M (U), 0N (V), 1N (W) and 2N (X). Scale bar: 50 μm.
Figure 9.
High magnification images of the immunohistochemical analysis of tau isoforms in 2 month-old wild-type mice reveals differences in subcellular localization.
Staining with pan-tau M antibody (A-C), 0N (D-F), 1N (G-I) and 2N (J-L). Close-up of the CA3 region (A,D,G,J), CA2 region (B,E,H,K) and nuclei in the CA3 region (C,F,I,L). Scale bar: 50 μm.
Figure 10.
Expression of murine tau in the hippocampus of 2-weeks old WT mice.
(A-D) Staining of the hippocampus with the new antibodies pan-tau M (A), 0N (B), 1N (C), and 2N (D) in red, counter-stained with Dako tau in green (E-H), merged images (I-L). (M-P) High magnification images of the hippocampal CA3 region using pan-tau M (M), 0N (N), 1N (O), and 2N (P). Scale bar: 50 μm.
Figure 11.
Expression of murine tau isoforms in the hippocampus of WT mice at day P0.
At this stage, 0N (0N3R) is the major isoform. (A-D) Staining with the pan-tau M antibody (A), 0N (B), 1N (C), and 2N (D). (E-H) Counter-staining with Dako tau. Scale bar: 50 μm.