Journal: Frontiers in Synaptic Neuroscience

Article Title: Selective Enrichment of Munc13-2 in Presynaptic Active Zones of Hippocampal Pyramidal Cells That Innervate mGluR1α Expressing Interneurons

doi: 10.3389/fnsyn.2021.773209

Figure Lengend Snippet: Munc13-2 immunolabeling is enriched on mGluR1α immunopositive dendrites. (A) Double immunolabeling for Munc13-2 (left, cyan) and mGluR1α (right, red) in the dorsal hippocampal CA1 region of the mouse (cartoon indicates the location of the region) shows similar distribution in the stratum oriens. Maximum intensity projection of six confocal images separated by 1 μm. (B) A dendritic segment of an mGluR1α immunopositive IN (white boxes on A ) is shown at a higher magnification, which is decorated by Munc13-2 immunopositive puncta. Maximum intensity projection of three confocal images separated by 1 μm. (C) 500 nm thick epoxy resin embedded section with preembedding immunolabeling for Munc13-2 (cyan) and mGluR1α (red) shows that Munc13-2 immunopositive puncta preferentially located on the small diameter of mGluR1α+ (distal) dendrites (dd), and mainly avoid the soma (s) and a proximal dendrite (pd) in the hippocampus of the mouse. Boxed area is enlarged on panel (D) . (D) Multiplexed postembedding immunolabeling carried out on the section shown in panel (G) . Munc13-2 immunopositive puncta marked by circles (representing ROIs for quantification) along the mGluR1α immunolabeled dendrite are immunopositive for Munc13-1, PSD95, AMPA receptors, Bassoon, Cav2.1,and Rim1/2 (all pseudo colored to green). Note that the intensity of Munc13-2 immunolabeling varies substantially. Alignment of sections after each round was based on mGluR1α immunolabeling (red). Numbers represent the labeling rounds during the multiplexed labeling. (E) All of the Munc13-2 immunopositive puncta contain PSD95 immunosignal. Their amount shows positive correlations (Spearman correlation r = 0.48 and 0.55, n = 40 in mouse #1 and n = 80 in mouse #2). (F) Correlations between the density of the Munc13-2 and Munc13-1 in individual AZs (each data point represents an AZ, n = 114 in mouse #1 and n = 80 in mouse #2; Spearman correlation r = 0.16 and 0.34). (G) mGluR1α IN targeting synapses have significantly larger (*) Munc13-1, AMPA receptors, Bassoon, Cav2.1 and Rim1/2 densities than those found in randomly selected glutamatergic synapses in the str. oriens ( p = 1.5 × 10 –5 , 5.5 × 10 –24 , 1.5 × 10 –4 , 6.6 × 10 –16 , 1.3 × 10 –5 , respectively, MW- U -test). Box plots represent median and 25/75 percentiles, square represent the mean value, whiskers represent SD. All immunolabelings were normalized to PSD95 intensity on panels (F,G) . (H) The Munc13-2 content of randomly selected synapses is only 4 ± 7% and 4 ± 10% (in two mice; p = 0 for both MW- U -test) of that of synapses on mGluR1α+ dendrites. (I–L) Munc13-2 immunolabeling (cyan) does not colocalize either with vesicular inhibitory amino acid transporter (VIAAT, red) ( I,J , n = 152 Munc13-2 and 222 VIAAT positive profiles in 2 mice) or with vesicular glutamate transporter-2 (vGluT2, red) (K,L) , n = 43 Munc13-2 and 33 vGluT2 positive profiles in 1 mouse). Single confocal images (I,K) . str. oriens, stratum oriens.

Article Snippet: Immunoglobulins were removed with a 5-min incubation in TBS containing 1% SDS (pH = 7.7) at 80 ° C. After 5 min of washing in TBST, new rounds of immunolabeling were performed using a guinea pig anti-PSD95 Ab (1:500, Synaptic Systems, Göttingen, Germany; Cat# 124014, RRID: AB_2619800 ), a guinea pig anti-panAMPA receptor Ab (1:100 Frontiers Cat#Af580; RRID: AB_257161 ), a chicken anti-Bassoon Ab (1:200 Synaptic Systems, Göttingen, Germany; Cat#141-016; RRID: AB_2661779 ), a guinea pig anti-Cav2.1 Ab (1:100 Synaptic Systems, Göttingen, Germany; Cat#152 205; RRID: AB_2619842 ), and finally with a guinea pig anti-Rim1/2 Ab (1:100 Synaptic Systems, Göttingen, Germany; Cat#140-205; RRID:AB_2631216 ) with the appropriate Alexa488-coupled secondary Abs (Alexa488-coupled donkey anti-guinea pig, 1:200; Jackson ImmunoResearch Laboratories, PA, United States; as above or Alexa488-coupled goat anti-chicken, 1:200; Jackson ImmunoResearch Laboratories, PA, United States, Cat# 103-545-155, RRID: AB_2337390 ).

Techniques: Immunolabeling, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Simple and Highly Efficient Detection of PSD95 Using a Nanobody and Its Recombinant Heavy-Chain Antibody Derivatives

doi: 10.3390/ijms24087294

Figure Lengend Snippet: Localization, domain structure and sequence of PSD95. ( A ) A sketch of an excitatory synapse. PSD95 is a major scaffold constituent of the post-synaptic density (PSD), localized juxtaposed to the synaptic vesicle release site. ( B ) Putative domain structure of PSD95: PDZ1/2/3, SH3, HOOK, and the non-catalytic Guanylate Kinase domain (GK). ( C ) Sequences of PDZ domains 1 and 2 of rat (Rt), human (Hu), and mouse (Ms) PSD95, as well as the most closely related proteins PSD93, SAP97, and SAP102 from rat. Within this sequence stretch, the rat and human PSD95 sequences are identical and differ by only two amino acids in the linker connecting PDZ1 and PDZ2 from the respective mouse sequence.

Article Snippet: Monoclonal anti-PSD95 antibody , MABN68 , Merck Millipore.

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Simple and Highly Efficient Detection of PSD95 Using a Nanobody and Its Recombinant Heavy-Chain Antibody Derivatives

doi: 10.3390/ijms24087294

Figure Lengend Snippet: NbPSD95 specifically recognizes PSD95. ( A ) A dilution series of FLAG-tagged NbPSD95 was applied to ELISA plates coated with indicated PDZ domains fused to E. coli maltose-binding protein (MBP). The detection was performed with an HRP-coupled anti-FLAG antibody. A significant signal could only be obtained for PSD95 PDZ1/2 . ( B ) COS-7 cells were transfected with the indicated PDZ-domains fused to EGFP and a mitochondrial targeting sequence. After fixation with 4% PFA, the cells were stained with NbPSD95 coupled to AZDye568 (structurally identical to AlexaFluor568). The co-localization of NbPSD95 with GFP required the presence of PSD95 PDZ1/2 . Scale bar: 20 µm. ( C ) Total brain lysates from a wild-type (WT) mouse or a PSD95 knock-out mouse (PSD95 KO) were analyzed by Western blot using r-hcAb comprising NbPSD95 and a Fc domain from either mouse IgG1 (MsIgG1 Fc; left) or rabbit IgG (RbIgG Fc; right). Actin served as a loading control. Both r-hcAbs readily detected PSD95 in the WT lysate, while no signal was obtained using the PSD95 KO lysate, indicating that the r-hcAbs exclusively recognize PSD95. ( D ) PFA-fixed hippocampal primary neuronal cultures from rats were stained with NbPSD95 coupled to AZDye568 (false color representation in red). Counterstaining was performed with an anti-GFAP nanobody (FluoTag-X2 anti-GFAP Atto488, false color representation in cyan). NbPSD95 produced the typical punctate staining on neuronal structures. Scale bar: 20 µm.

Article Snippet: Monoclonal anti-PSD95 antibody , MABN68 , Merck Millipore.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Transfection, Sequencing, Staining, Knock-Out, Western Blot, Produced

Journal: International Journal of Molecular Sciences

Article Title: Simple and Highly Efficient Detection of PSD95 Using a Nanobody and Its Recombinant Heavy-Chain Antibody Derivatives

doi: 10.3390/ijms24087294

Figure Lengend Snippet: Comparison of NbPSD95 with an anti-PSD95 r-hcAb and a conventional antibody. Primary hippocampal neurons were stained with NbPSD95 directly labeled with AbberiorStar635P (nanobody), with an anti-PSD95 r-hcAb harboring a mouse IgG2 Fc domain (r-hcAb) or an established mouse monoclonal IgG2 antibody (antibody), all at a concentration of 14 nM ( A ) or 1 nM ( B ). Identical concentrations of FluoTag-X2 anti-Mouse IgG2 coupled to AbberiorStar635P were used as secondary reagents for the detection of both the r-hcAb and the mouse monoclonal antibody. Merged figures show the respective anti-PSD95 signal (red) together with counter-stainings performed with FluoTag-X2 anti-GFAP conjugated to Atto488 (grey) and FluoTag-X2 anti-Syt1 (synaptotagmin 1) conjugated to AZDye568 (cyan). Confocal images were acquired with equal settings for each channel and images equally scaled to allow a direct comparison. Separate channels are shown in .

Article Snippet: Monoclonal anti-PSD95 antibody , MABN68 , Merck Millipore.

Techniques: Staining, Labeling, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Simple and Highly Efficient Detection of PSD95 Using a Nanobody and Its Recombinant Heavy-Chain Antibody Derivatives

doi: 10.3390/ijms24087294

Figure Lengend Snippet: Confocal and STED imaging on primary rat hippocampal neurons. Primary hippocampal culture neurons were stained using NbPSD95 (FluoTag-X2 anti-PSD95, red) conjugated to AbberiorStar635P and imaged using Confocal and STED microscopy. For counterstaining, the pre-synaptic protein Synaptotagmin 1 (Syt1; cyan) and glia marker GFAP (grey) were imaged in confocal mode for reference. Four selected regions denoted by white squares are magnified, displaying conventional lateral PSD shapes (asterisks) as well as so-called perforated synapses (arrowheads) only visible under STED microscopy.

Article Snippet: Monoclonal anti-PSD95 antibody , MABN68 , Merck Millipore.

Techniques: Imaging, Staining, Microscopy, Marker

Journal: International Journal of Molecular Sciences

Article Title: Simple and Highly Efficient Detection of PSD95 Using a Nanobody and Its Recombinant Heavy-Chain Antibody Derivatives

doi: 10.3390/ijms24087294

Figure Lengend Snippet: Immunohistochemistry using NbPSD95 on formalin-fixed brain slices. Mouse (left) and rat (right) brains were fixed in 4% formaldehyde for 24 h and cut into 50 µm thick sections using a vibratome. Sections were stained with Sulfo-Cy3-conjugated NbPSD95 (red) and DAPI (blue). Without antigen retrieval, the nanobody reveals PSD95-positive synapses in the mouse and rat brains. ( A ) Epifluorescence imaging of the mouse and rat hippocampus shows the expected intensity pattern with strong and dense staining in the stratum oriens and stratum radiatum of CA1. ( B – D ) Higher magnification images show numerous synaptic dots in the hippocampal regions of CA1 ( B ) and CA3 ( C ) or in the cortex ( D ). The staining pattern suggests a high specificity for NbPSD95 towards excitatory synapses. In all regions, NbPSD95 works comparably well in mouse and rat specimens.

Article Snippet: Monoclonal anti-PSD95 antibody , MABN68 , Merck Millipore.

Techniques: Immunohistochemistry, Staining, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Simple and Highly Efficient Detection of PSD95 Using a Nanobody and Its Recombinant Heavy-Chain Antibody Derivatives

doi: 10.3390/ijms24087294

Figure Lengend Snippet: IHC-P using an anti-PSD95 r-hcAb. Chromogenic IHC staining of an anti-PSD95 r-hcAb in formalin-fixed paraffin-embedded (FFPE) mouse (left) and rat (right) tissue sections using 3,3′-Diaminobenzidin (DAB; brown staining). Hematoxylin provides blue nuclear staining. ( A ) Immunohistochemistry using the rabbit anti-PSD95 r-hcAb reveals the characteristic distribution of PSD95 in coronal sections of mouse and rat hippocampi. All regions of the hippocampus are labeled. However, DAB staining is more intense in the pyramidal cell layer of the CA1 area and in the molecular layer of the dentate gyrus (DG). ( B ) High-magnification views of mouse and rat FFPE cerebellum sections. Strong PSD95 staining is detected within the basket cell terminal pinceaux (arrows). Purkinje cells (P) and neurons in the granule layer are not stained. ( C ) In FFPE sections of mouse and rat retina, very intense DAB staining is present in the outer plexiform layer (OPL). Moderate punctate staining is observed in the inner plexiform layer (IPL). The inner and outer nuclear layers (INL and ONL) are not stained.

Article Snippet: Monoclonal anti-PSD95 antibody , MABN68 , Merck Millipore.

Techniques: Paraffin-embedded Immunohistochemistry, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Staining, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Simple and Highly Efficient Detection of PSD95 Using a Nanobody and Its Recombinant Heavy-Chain Antibody Derivatives

doi: 10.3390/ijms24087294

Figure Lengend Snippet: Antibodies.

Article Snippet: Monoclonal anti-PSD95 antibody , MABN68 , Merck Millipore.

Techniques: Recombinant, Avidin-Biotin Assay, Plasmid Preparation