Journal: Journal of Neuroscience Research

Article Title: Lanthionine Ketimine Ethyl Ester Induces Proliferation and Maturation and Regulates Calcium Flux in Primary Mouse Oligodendrocyte Progenitor Cells

doi: 10.1002/jnr.70061

Figure Lengend Snippet: LKE increases OPC maturation. Primary mouse OPCs were incubated with 50 μM LKE for 2 or 4 days, after which the cells were fixed and processed for immunocytochemical staining with the indicated antibodies. Representative images are shown for (A) Olig2; (B) CC1; (C) Olig2 plus CC1; (D) NG2; (E) PLP; and (F) Caspase‐3. The average number of cells per field of view is shown. The % Olig2+/Ki67+ cells is the % of the Olig2+ cells that co‐express CC1. ** p < 0.05; *** p < 0.005 versus same day control cells, 1‐way ANOVA, Tukey's post hoc comparisons. The scale bar shown in panel F is 90 μm and is the same for all panels.

Article Snippet: The primary antibodies used for immunocytochemistry were against caspase‐3 (mouse; 1:400; Cell Signaling, 96,615), the anti‐adenomatous polyposis coli (APC) clone CC1 (mouse; 1:300; EMD Millipore, OP80), the marker of proliferation Ki67 (mouse; 1:200; BD Bio‐sciences, AB_393778), the proteoglycan neuron‐glial antigen 2 (NG2, rabbit; 1:400; END Millipore, AB_5320), the OLG transcription factor Olig2 (rabbit; 1:500; EMD Millipore, AB_9610), and proteolipid protein (PLP, rat; 1:250, AA3‐PLP/DM20).

Techniques: Incubation, Staining, Control

Journal: ASN NEURO

Article Title: Analysis of Oligodendrocyte Lineage Cell Progression with Cre-Mediated RiboTag Reporter Lines

doi: 10.1080/17590914.2025.2513885

Figure Lengend Snippet: Generation of constitutive NG2 Cre :Rpl22 HA mice to investigate oligodendrocyte lineage cells. a) Schematic outlining the genetic strategy for crossing RiboTag (RPL22 HA ) mice with constitutive NG2 Cre mice, where Cre recombinase expression is under the control of the Cspg4 promoter. Mice expressing both RPL22 HA and Cre express an HA tag on cells expressing Cspg4 (NG2), including oligodendrocyte lineage cells (OLCs). b) PCR products using oligonucleotides that amplify the Cre-containing sequence (left) and the loxp-containing intron in the Rpl22 gene (right). The Cre-positive control band is 300 bp, while the mutant Cre product is 100 bp. The WT Rpl22 product is 260 bp, while the mutant product is 290 bp. c) Western analysis of wild-type RPL22 protein or RPL22 HA in control Cre, littermate control, and double mutant HA-expressing mice. Blots were probed with anti-HA antibodies. RPL22 HA results in a 23 kDa protein, while the native RPL22 is a 15 kDa protein. d) Western blots after immunoprecipitation of spinal cord homogenates using anti-HA or control IgG antibodies, demonstrating specific immunoprecipitation of RPL22 HA protein in double mutant HA-expressing mice. e) Immunofluorescence reporter studies demonstrate that HA-labeling is only displayed in NG2 Cre+ :Rpl22 HA mice (right), while there is no labeling in NG2 Cre+ :Rpl22 WT (left) or NG2 Cre- :Rpl22 HA mice (center). Scale bar = 50 µm, 25 µm sections . f) Visualization of co-labeling of the HA-tag and OLIG2+ OLCs in the corpus callosum (top) and spinal cord white matter (bottom). Quantification of the co-labeling (including the cerebellum, Supplementary Fig. 1a ) demonstrates 93.4% of OLIG2+ cells are HA+ (bottom right). N = 2, 12.5 µm sections, scale bar = 50 µm (top), 20 µm (bottom). g) Visualization demonstrating co-labeling of the HA-tag with PDGFRA+, NG2+ oligodendrocyte precursor cells (OPCs) in the cerebral cortex (left) and spinal cord (right). Scale bar = 20 µm, arrows denote co-labeling. h) Visualization demonstrating co-labeling of the HA-tag with PDGFRA+ OPCs and CC1+ mature oligodendrocytes (mOLs). i–j ) Quantification of the co-labeling demonstrates 75.36% of PDGFRA+ OPCs and 82.71% of CC1+ mOLs are also HA+ ( i ), while 88.79% of HA+ cells are either PDGFRA+ or CC1+ ( j ). N = 2, scale bar = 20 µm, white arrows denote PDGFRA+/HA+ co-labeling and yellow arrows denote CC1+/HA+ co-labeling, SCWM = spinal cord white matter. All quantifications are presented as mean +/− SEM.

Article Snippet: APC (CC1) , Mouse , 1:100 , Calbiochem (LSBio).

Techniques: Expressing, Control, Sequencing, Positive Control, Mutagenesis, Western Blot, Immunoprecipitation, Immunofluorescence, Labeling

Journal: ASN NEURO

Article Title: Analysis of Oligodendrocyte Lineage Cell Progression with Cre-Mediated RiboTag Reporter Lines

doi: 10.1080/17590914.2025.2513885

Figure Lengend Snippet: NG2 Cre :Rpl22 HA enables tracking of oligodendrocyte lineage cells following focal demyelination. a) Schematic of the lysolecithin focal demyelination model where 1 µL of 1% LPC is injected into the ventral white matter (left) creating a focal demyelinated area (center). The demyelinated area is visualized by an accumulation of nucleated cells which can be labeled and identified with Dapi staining (right). Dashed line represents the borders of the lesioned area. b) Representative immunofluorescence (IF) images demonstrating both the tracing of a lesion using accumulated nucleated cells as a lesion identifier overlayed with OLIG2 (left) and the colocalization of the Rpl22 HA tag with OLIG2+ cells inside the lesion (right). Inset is zoomed in image of the lesion. c) Schematic of the lysolecithin lesion time course in the spinal cord, highlighting reproducible and selected timepoints coinciding with OPC recruitment (3–5 days post lesion, dpl), OPC differentiation (10–14 dpl), and oligodendrocyte maturation (20-30 dpl). d) Visualization of the population of HA+ and OLIG2+ cells in constitutive NG2 Cre :Rpl22 HA mouse lesions at 7 dpl (left), 14 dpl (center) and 21 dpl (right). Scale bar = 50 µm. Insets show zoomed in lesioned area. e) Quantification of OLIG2+, HA+, and double positive cells throughout the three timepoints and the percentage of colocalization. Quantification of the colocalization ratio of HA+/OLIG2+ cells show ∼85% throughout all three timepoints, with no significant difference between any of the three timepoints. N = 2–3. f) Visualization demonstrating co-labeling of the HA-tag with PDGFRA+ OPCs and CC1+ mature oligodendrocytes at 21dpl (left), and quantification of the co-labeling demonstrates 82.42% of PDGFRA+ OPCs and 84.34% of CC1+ mOLs are also HA+ (right). Scale bar = 50 µm, white arrows denote PDGFRA+/HA+ co-labeling, yellow arrows denote CC1+/HA+ co-labeling. Inset is a higher magnification of the boxed area of interest, inset scale bar = 20 µm. All quantifications are presented as mean +/− SEM.

Article Snippet: APC (CC1) , Mouse , 1:100 , Calbiochem (LSBio).

Techniques: Injection, Labeling, Staining, Immunofluorescence

Journal: ASN NEURO

Article Title: Analysis of Oligodendrocyte Lineage Cell Progression with Cre-Mediated RiboTag Reporter Lines

doi: 10.1080/17590914.2025.2513885

Figure Lengend Snippet: Analysis of RiboTag labeling in inducible Pdgfra CreERT :Rpl22 HA and Plp CreERT :Rpl22 HA Mice to Label OLCs . a) Schematic outlining the genetic strategy for crossing RPL22 HA mice with tamoxifen inducible Pdgfra CreERT and Plp CreERT mice. Mice expressing both RPL22 HA and Cre express an HA tag on targeted OLC populations after the addition of 4-hydroxytamoxifen (4daily i.p. injections, 4 mg/kg, 250 µL/injection, 1 mL total). b) Representative immunofluorescence (IF) of RPL22 HA co-labeling with OLIG2+ OLCs in Pdgfra CreERT :Rpl22 HA mice (top) and Plp CreERT :Rpl22 HA (bottom) in the corpus callosum (top right, scale bar = 50 µm) and spinal cord white matter (left, scale bar = 20 µm). Colocalization quantification across regions of interest indicates that 37.3% of the OLIG2+ population is RPL22 HA labeled in Pdgfra CreERT :Rpl22 HA and 82.0% in Plp CreERT :Rpl22 HA (bottom right). N = 2, arrows denote co-labeled cells. c-d) Representative IF imaging ofPdgfra CreERT :Rpl22 HA mouse spinal cord indicating Rpl22 HA reporter co-labeling with PDGFRA+ OPCs and CC1+ mOLs. ( d ) is a higher magnification image of the outlined region in ( c ). Scale bar = 50 µm ( c ), 20 µm ( d ), white arrows denote PDGFRA+/HA+ OPCs, and yellow arrows denote CC1+/HA+ mOLs, GM = gray matter, WM = white matter, yellow dotted line traces the GM. e) Quantification of co-labeled PDGFRA+ or CC1+ cells with HA+ over the total PDGFRA+ or CC1+ cells demonstrates near all PDGFRA+ OPCs express HA while 18% of CC1+ mOLS express HA. SCWM = spinal cord white matter. f) Quantification of HA-expressing cells indicates 94.33% of HA+ cells are CC1 + mOLS or PDGFRA+ OPCs. N = 3. g–h) Representative IF imaging of Plp CreERT :Rpl22 HA mouse spinal cord indicating co-labeling with CC1+ mOLs and a lack of reporter labeling with PDGFRA+ OPCs. ( h ) is a higher magnification image of the white outlined region in ( g ). Scale bar = 50 µm ( g ), 20 µm ( h ), arrows denote CC1+/HA+ cells, GM = gray matter, WM = white matter, yellow dotted line traces the GM. i) Quantification of co-labeled PDGFRA+ or CC1+ cells with HA+ over the total PDGFRA+ or CC1+ cells demonstrates almost no PDGFRA+ OPCs express HA, while 87.0% of CC1+ mOLs also express HA. SCWM = spinal cord white matter. j) Quantification of HA-expressing cells indicates 93.95% of HA+ cells are CC1+ mOLs. N = 2. All quantifications are presented as mean +/− SEM.

Article Snippet: APC (CC1) , Mouse , 1:100 , Calbiochem (LSBio).

Techniques: Labeling, Expressing, Injection, Immunofluorescence, Imaging

Journal: ASN NEURO

Article Title: Analysis of Oligodendrocyte Lineage Cell Progression with Cre-Mediated RiboTag Reporter Lines

doi: 10.1080/17590914.2025.2513885

Figure Lengend Snippet: Inducible Pdgfra CreERT :Rpl22 HA and Plp CreERT :Rpl22 HA mice enable labeling of OLCs following focal demyelination. a) Immunofluorescence (IF) imaging demonstrating Rpl22 HA co-labeling with OLIG2+ OLCs inside the lesion at 5dpl in Pdgfra CreERT :Rpl22 HA mice. Scale bar = 50 µm, arrows denote co-labeled HA+/OLIG2+ cells, outline denotes lesioned area. b) IF demonstrating an almost complete lack of Rpl22 HA in the lesion at 5dpl in Plp CreERT :Rpl22 HA mice. Scale bar = 50 µm, arrows denote co-labeled HA+/OLIG2+ cells, outline denotes lesioned area. c) IF demonstrating Rpl22 HA reporter co-labeling with CC1+ mOLs at the lesion edge of Plp CreERT :Rpl22 HA . (top). The yellow dotted line indicates lesion boundary, LE = lesion edge. Quantification of HA+/CC1+ mOL density at the lesion edge and nonlesioned spinal cord white matter (bottom), indicating a higher density at the lesion edge. N = 2. NLWM = nonlesioned white matter. Quantifications are presented as mean +/ − SEM. d-e) No Rpl22 HA co-labeling was observed with GFAP+ astrocytes (left) or IBA1+ microglia (right) in either Pdgfra CreERT :Rpl22 HA ( d ) or Plp CreERT :Rpl22 HA ( e ) mice at 5dpl. f) IF imaging demonstrating HA+ reporter co-labeling with PDGFRA+/NG2+ OPCs in Pdgfra CreERT :Rpl22 HA at 5dpl. Inset demonstrates Hoechst staining. Higher magnification split channels of boxed region of interest are shown to the right, arrows denote HA+/PDGFRA+/NG2+ OPCs, scale bars = 50 µm (left) and 20 µm (right). g) IF imaging demonstrating HA+ reporter co-labeling with CC1+ mOLs and rare PDGFRA+ OPCs in Plp CreERT :Rpl22 HA at 5dpl. Inset demonstrates Hoechst staining. Higher magnification split channels of boxed region of interest are shown to the right, white arrow denotes PDGFRA+/HA+ OPC, yellow arrow denotes CC1+/HA+ mOL, scale bars = 50 µm (left) and 20 µm (right).

Article Snippet: APC (CC1) , Mouse , 1:100 , Calbiochem (LSBio).

Techniques: Labeling, Immunofluorescence, Imaging, Staining

Journal: ASN NEURO

Article Title: Analysis of Oligodendrocyte Lineage Cell Progression with Cre-Mediated RiboTag Reporter Lines

doi: 10.1080/17590914.2025.2513885

Figure Lengend Snippet: Following focal demyelination, Pdgfra CreERT :Rpl22 HA and Plp CreERT :Rpl22 HA mice display distinct reporting patterns. a) Immunofluorescence (IF) images demonstrating co-labeling of Rpl22 HA with OLIG2+ OLCs at 10dpl (left) and 20dpl (right) in Pdgfra CreERT :Rpl22 HA mice. Scale bar = 50 µm. b) Quantifications indicating expected increasing OLIG2+ and HA+ cells in the lesion and the co-labeling of lesion-associated OLIG2+ OLCs. N = 1–2. c) IF images showing Rpl22 HA co-labeling with NKX2.2+ differentiating OLCs at 10dpl (left), and CC1+ mOLs at 20dpl (right) in Pdgfra CreERT :Rpl22 HA . Scale bar = 50 µm. d) Quantification of the HA+/NKX2.2+ co-labeling at 5 and 10dpl (top), and HA+/CC1+ co-labeling across all observed timepoints (bottom), indicating reporter labeling of recruited OPCs as they generate new mOLs. N = 1–2. e) IF images demonstrating Rpl22 HA reporter co-labeling with OLIG2+ OLCs at 10 and 20dpl (left) in Plp CreERT :Rpl22 HA . Scale bar = 50 µm, outline denotes lesioned area. Quantification of HA+/Olig2+ co-labeling indicates that the percentage of OLIG2+ cells that are also HA+ increases over time to 35.70% by 20dpl. N = 2. f) IF images demonstrating HA+ co-labeling with CC1+ mOLs at 10 and 20dpl (left) in Plp CreERT :Rpl22 HA . Scale bar = 50 µm, outline denotes lesioned area. Quantification of HA+CC1+ co-labeling indicates that the majority of CC1+ mOLs found in the lesion are co-labeled with HA by 20dpl (62.00%). N = 2. g) No co-labeling was observed between the Rpl22 HA reporter and PDGFRA+ OPCs in lesions in Plp CreERT :Rpl22 HA mice at 10dpl. White arrows denote PDGFRA+ cells., outline denotes lesioned area, right is higher magnification of the boxed region of interest. Scale bars = 50 µm (left) and 20 µm (right). h) IF imaging demonstrating the HA+ reporter patterning and accumulation at 5, 10, and 20dpl. Scale bar = 50 µm. i) Quantifications of the accumulation of HA+ cells (far left), Olig2+ cells (middle left), HA+,Olig2+ cells (middle right) over all observed timepoints. N = 2. Quantification of HA+ cells that are also OLIG2+ (far right) indicates that in Plp CreERT :Rpl22 HA mice ∼60% of HA+ cells are of the oligodendrocyte lineage. N = 2. All quantifications are presented as mean +/− SEM.

Article Snippet: APC (CC1) , Mouse , 1:100 , Calbiochem (LSBio).

Techniques: Immunofluorescence, Labeling, Imaging

Journal: Cell reports

Article Title: Disruption of neuronal RHEB signaling impairs oligodendrocyte differentiation and myelination through mTORC1-DLK1 axis

doi: 10.1016/j.celrep.2023.112801

Figure Lengend Snippet: (A and B) Western blots and quantification showing the expression of myelin proteins (MBP, TMEM10, MOBP) of the cortex of 4-week-old Rheb f/f ;CamKII-Cre ( Rheb cKO), Rheb f/f ;Dlk1 f/f ;CamKII-Cre ( Rheb / Dlk1 dcKO), and control ( Rheb f/f , Dlk1 f/f , Rheb f/f ; Dlk1 f/f ; Ctrl) mice. n = 6 mice for each genotype. Error bars represent mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, Tukey’s multiple comparisons test, combined with one-way ANOVA analysis). (C) Immunostaining of MBP of the cortex of 4-week-old Rheb f/f ;CamKII-Cre , Rheb f/f ;Dlk1 f/f ;CamKII-Cre , and control ( Rheb f/f ; Dlk1 f/f ) mice. cc, corpus callosum. n = 3 mice for each genotype. Scale bar: 200 μm. (D–F) Electron micrographs (D) and quantification showing the myelinated axons (E) and g-ratio (F) of the cortex of 6-week-old Rheb f/f ;CamKII-Cre , Rheb f/f ;Dlk1 f/f ;CamKII-Cre (dcKO), and control ( Rheb f/f , Rheb f/f ; Dlk1 f/+ ) mice. Red asterisks indicate myelinated axons. n = 3 Rheb cKO, 3 Rheb/Dlk1 dcKO and 5 control mice. Error bars represent mean ± SD (**p < 0.01, ****p < 0.001, Tukey’s multiple comparisons test, combined with one-way ANOVA analysis). Scale bar: 1 μm. (G and H) Immunostaining of OLIG2 (red) and CC1 (green) and quantification showing the cell density of postmitotic oligodendrocytes (H) of the cortex of 4-week-old Rheb f/f ;CamKII-Cre , Rheb f/f ;Dlk1 f/f ;CamKII-Cre (dcKO), and control ( Rheb f/f , Rheb f/f ; Dlk1 f/f ) mice. n = 4 mice for each genotype. Error bars represent mean ± SD (*p < 0.05, **p < 0.01, ns means no statistical significance, Tukey’s multiple comparisons test, combined with one-way ANOVA analysis). Scale bar: 50 μm. See also .

Article Snippet: Mouse monoclonal anti-APC (CC1) , Millipore , Cat# OP80;RRID: AB_2057371.

Techniques: Western Blot, Expressing, Control, Immunostaining

Journal: Cell reports

Article Title: Disruption of neuronal RHEB signaling impairs oligodendrocyte differentiation and myelination through mTORC1-DLK1 axis

doi: 10.1016/j.celrep.2023.112801

Figure Lengend Snippet: (A and B) Western blots and quantification showing the expression of HA-tagged DLK1 protein and myelin proteins (MBP, TMEM10, MOBP, MOG) of the cortex of 3-week-old mDlk1 k/+ ;CamKII-Cre ( mDlk1 Tg) and control (wild-type, mDlk1 k/+ , and CamKII-Cre , Ctrl) mice. n = 5 pairs of mice. Error bars represent mean ± SD (***p < 0.001, two-tailed unpaired t test). (C and D) MOG immunostaining (C) and Black Gold staining (D) of the cortex of 3-week-old mDlk1 k/+ ;CamKII-Cre and control (wild-type and CamKII-Cre ) mice. cc, corpus callosum. n = 3 pairs of mice. Scale bar: 100 μm. (E and F) Electron micrographs and quantification showing the myelinated axons of the cortex of 6-week-old mDlk1 k/+ ; CamKII-Cre and control (wild-type, mDlk1 k/+ , and CamKII-Cre ) mice. Red asterisks indicate myelinated axons. n = 3 pairs of mice. Error bars represent mean ± SD (**p < 0.01, two-tailed unpaired t test). Scale bar: 1 μm. (G and H) Immunostaining of OLIG2 (red) and CC1 (green) and quantification showing the cell density of oligodendrocytes (OLIG2 + CC1 + ) of the cortex of 3-week-old mDlk1 k/+ ;CamKII-Cre and control (wild-type and CamKII-Cre ) mice. n = 4 pairs of mice. Error bars represent mean ± SD (**p < 0.01, two-tailed unpaired t test). Scale bar: 50 μm. (I and J) Immunostaining of BrdU (red) and OLIG2 (green) and quantification showing the cell density of BrdU-labeled OPC (BrdU + OLIG2 + ) of the cortex after BrdU injection of P10 mDlk1 k/+ ;CamKII-Cre and control (wild-type and CamKII-Cre ) mice. n = 4 pairs of mice. Error bars represent mean ± SD (ns means no statistical significance, two-tailed unpaired t test). Scale bar: 50 μm. (K and L) Immunostaining of BrdU (red) and CC1 (green) and quantification showing the cell density of BrdU-labeled oligodendrocytes (BrdU + CC1 + ) of the cortex after BrdU injection of 3-week-old mDlk1 k/+ ;CamKII-Cre and control (wild-type, mDlk1 k/+ , and CamKII-Cre ) mice. n = 4 pairs of mice. Error bars represent mean ± SD (**p < 0.01, two-tailed unpaired t test). Scale bar: 50 μm. See also .

Article Snippet: Mouse monoclonal anti-APC (CC1) , Millipore , Cat# OP80;RRID: AB_2057371.

Techniques: Western Blot, Expressing, Control, Two Tailed Test, Immunostaining, Staining, Labeling, Injection

Journal: Cell reports

Article Title: Disruption of neuronal RHEB signaling impairs oligodendrocyte differentiation and myelination through mTORC1-DLK1 axis

doi: 10.1016/j.celrep.2023.112801

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-APC (CC1) , Millipore , Cat# OP80;RRID: AB_2057371.

Techniques: Recombinant, SYBR Green Assay, Membrane, Bicinchoninic Acid Protein Assay, Reverse Transcription, Western Blot, Multiplex Assay, Software

Journal: Frontiers in Cellular Neuroscience

Article Title: Cryopreservation of primary neonatal rat oligodendrocytes and recapitulation of in vitro oligodendrocyte characteristics

doi: 10.3389/fncel.2024.1520992

Figure Lengend Snippet: Differentiation of cryopreserved OPCs into mature OLs. (A) Representative images of control and cryopreserved OPCs after 2 days of stabilization, followed by 4 days of differentiation into mature OLs. Immunostaining for MBP, CC1. (B) Graph of mature OL proportions, measured as MBP + DAPI + mature OLs/DAPI + total cells ( n = 4 biologically independent experiments per group, with three experimental groups. For each group, 10 images were analyzed, with approximately 75 DAPI + cells per image. Statistical analysis was performed using a two-sided Kruskal–Wallis test. Data are presented as mean ± SEM). (C) Graph of mature OL proportions, measured as CC1 + DAPI + mature OLs/DAPI + total cells ( n = 4 biologically independent experiments per group, with three experimental groups. For each group, 10 images were analyzed, with approximately 75 DAPI + cells per image. Statistical analysis was performed using a two-sided Kruskal–Wallis test. Data are presented as mean ± SEM). OPC, oligodendrocyte progenitor cell; OL, oligodendrocyte; MBP, myelin basic protein; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; T3, thyroid hormone T3.

Article Snippet: Following a PBS wash, the cells were incubated overnight (~16 h) at 4°C with the following primary antibodies suspended in the blocking solution: anti-NG2 (1:200, Merck Millipore, #AB5320), anti-myelin basic protein (MBP) (1:500, Abcam, #AB7349), anti-Olig2 (1:200, Merck Millipore, #MABN50), anti-adenomatous polyposis coli (APC-CC1) (1:200, Merck Millipore, #OP80).

Techniques: Control, Immunostaining, Derivative Assay