Figure S3. Microglia activity... | Rockefeller University Press

$Microglia activity categorization, densities in unaffected brain regions, isolation of microglia, and measures of engulfment of P2X2+terminals and CD68+. (A) Examples of the four categories in microglia. The top panels show examples of microglia categorized into each of the four categories. The bottom panels show the CD68+ label for each microglia example. Scale bars = 10 µm. Images of single microglia were extracted from confocal stack images of multiple microglia through Imaris software (Oxford Instruments). A black background was used to allow visualization of CD68+ labeling (white). Suitable examples of microglia representing category 5 could not be found in tissue imaged at higher magnifications used to provide clear images because they represent <1% of the total microglia. (B and C) Microglia densities of control and E3–E12 diet mice in the hypoglossal nucleus (B) and dLGN (control—P15, n = 4; adult, n = 4; E3–E12 diet—P15, n = 5; adult, n = 4; C). Note that measures were taken from P7 mice for lateral geniculate nucleus measurements to match the normal pruning period of this circuit (Schafer et al., 2012; Stevens et al., 2007). (D and E) Gating strategy (D) and representative plots (E) of microglia purity before RNA-seq. (F) Percent of CD45+ cells that are also CD11b+ in the fraction used for analyses (+ fraction) and negative fraction in samples from P15 controls (n = 3) and E3–E12 diet mice (n = 3). (G) Percent of P2X2+ label engulfed by microglia normalized to the volume of the brain sections analyzed in P15 controls and E3–E12 diet mice (n = 6, 7, respectively) and adult controls and E3–E12 diet mice (n = 7, 6, respectively). (H) Percent of large microglia occupied by CD68+ label in P15 controls and E3–E12 diet mice (n = 7, 7, respectively) and adult controls and E3–E12 diet mice (n = 7, 6, respectively). (I) Percent of CD68+ in microglia normalized to the volume of the brain sections analyzed in P15 controls and E3–E12 diet mice (n = 7, 7, respectively) and adult controls and E3–E12 diet mice (n = 7, 6, respectively). (J) Percent of P2X2+ colocalized with CD68+ in activated microglia in P15 controls and E3–E12 diet mice (n = 6, 7, respectively) and adult controls and E3–E12 diet mice (n = 7, 6, respectively). For all dietary group comparisons, statistical comparisons were multiple, unpaired t tests using the Holm–Sidak method for multiple comparisons. * denotes P < 0.05. (F) +Fraction versus −Fraction for controls, P = 0.0001; +Fraction versus −Fraction for E3–E12 mice, P = 0.0001. (G) P22 controls versus P22 E3–E12 diet, P = 0.007. (H) Adult controls versus adult E3–E12 diet, P = 0.008. (I) P22 control versus E3–E12 diet, P = 0.02. One observation/animal was obtained and analyzed for data shown in B, C, and G–J. For F, NSTs were pooled from six littermates with respect to treatment so that each biological replicate is representative of one litter. In total, three litters were used from control and from E3–E12 diet mice. Data in B, C, and F–J are shown as mean ± SEM.$

Figure S3.

Microglia activity categorization, densities in unaffected brain regions, isolation of microglia, and measures of engulfment of P2X2 ⁺ terminals and CD68 ⁺ . (A) Examples of the four categories in microglia. The top panels show examples of microglia categorized into each of the four categories. The bottom panels show the CD68⁺ label for each microglia example. Scale bars = 10 µm. Images of single microglia were extracted from confocal stack images of multiple microglia through Imaris software (Oxford Instruments). A black background was used to allow visualization of CD68⁺ labeling (white). Suitable examples of microglia representing category 5 could not be found in tissue imaged at higher magnifications used to provide clear images because they represent <1% of the total microglia. (B and C) Microglia densities of control and E3–E12 diet mice in the hypoglossal nucleus (B) and dLGN (control—P15, n = 4; adult, n = 4; E3–E12 diet—P15, n = 5; adult, n = 4; C). Note that measures were taken from P7 mice for lateral geniculate nucleus measurements to match the normal pruning period of this circuit (Schafer et al., 2012; Stevens et al., 2007). (D and E) Gating strategy (D) and representative plots (E) of microglia purity before RNA-seq. (F) Percent of CD45⁺ cells that are also CD11b⁺ in the fraction used for analyses (+ fraction) and negative fraction in samples from P15 controls (n = 3) and E3–E12 diet mice (n = 3). (G) Percent of P2X2⁺ label engulfed by microglia normalized to the volume of the brain sections analyzed in P15 controls and E3–E12 diet mice (n = 6, 7, respectively) and adult controls and E3–E12 diet mice (n = 7, 6, respectively). (H) Percent of large microglia occupied by CD68⁺ label in P15 controls and E3–E12 diet mice (n = 7, 7, respectively) and adult controls and E3–E12 diet mice (n = 7, 6, respectively). (I) Percent of CD68⁺ in microglia normalized to the volume of the brain sections analyzed in P15 controls and E3–E12 diet mice (n = 7, 7, respectively) and adult controls and E3–E12 diet mice (n = 7, 6, respectively). (J) Percent of P2X2⁺ colocalized with CD68⁺ in activated microglia in P15 controls and E3–E12 diet mice (n = 6, 7, respectively) and adult controls and E3–E12 diet mice (n = 7, 6, respectively). For all dietary group comparisons, statistical comparisons were multiple, unpaired t tests using the Holm–Sidak method for multiple comparisons. * denotes P < 0.05. (F) +Fraction versus −Fraction for controls, P = 0.0001; +Fraction versus −Fraction for E3–E12 mice, P = 0.0001. (G) P22 controls versus P22 E3–E12 diet, P = 0.007. (H) Adult controls versus adult E3–E12 diet, P = 0.008. (I) P22 control versus E3–E12 diet, P = 0.02. One observation/animal was obtained and analyzed for data shown in B, C, and G–J. For F, NSTs were pooled from six littermates with respect to treatment so that each biological replicate is representative of one litter. In total, three litters were used from control and from E3–E12 diet mice. Data in B, C, and F–J are shown as mean ± SEM.

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