Murine Esophagus Expresses Glial-Derived Central Nervous System Antigens
Abstract
:1. Introduction
2. Results
2.1. The Esophagus Expresses CNS Typical Markers at the mRNA-Level
2.2. Distribution of MBP, PLP and GFAP in the Cerebellum
2.3. Glial Cells in Neuromuscular Junctions of the Tibialis Anterior Muscle
2.4. Glial Cells in Neuromuscular Junctions of the Esophagus
2.5. Transgenic Mice Reveal the Presence of Different Types of Enteric Glial Cells in the Esophagus
3. Discussion
3.1. Presence of Glial Cell Specific Markers in the Esophageal Neuromuscular Junction
3.2. Different Degrees of Myelination in the Myenteric Plexus
3.3. Differentiation of Esophageal Enteric Glial Cells by the Use of Glial Markers
3.4. Enteric Glial Cells as an Immunological Target
4. Materials and Methods
4.1. Mice
4.2. RT-PCR
4.2.1. Tissue Preparation
4.2.2. Processing
4.3. Immunohistochemistry
4.3.1. Tissue Preparation and Fixation
4.3.2. Frozen Sections
4.3.3. Whole Mounts
4.3.4. Control Experiments
4.4. Image Aquisition
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
BSA | Bovine serum albumin |
CALB | Calbindin (D28k) |
cDNA | Complementary DNA |
ChAT | Choline Acetyltransferase |
CNS | Central nervous system |
DEPC | Diethyl pyrocarbonate |
DsRed | Discosoma sp. red fluorescent protein |
EAE | Experimental autoimmune encephalomyelitis |
EDTA | Ethylenediaminetetraacetic acid |
EGC | Enteric glial cell |
EGFP | Enhanced green fluorescent protein |
ENS | Enteric nervous system |
GFAP | Glial fibrillary acidic protein |
GFP | Green fluorescent protein |
MAG | Myelin-associated glycoprotein (also: Siglec-4) |
MBP | Myelin basic protein |
MOG | Myelin oligodendrocyte glycoprotein |
MS | Multiple sclerosis |
NMJ | Neuromuscular junction |
OSP | Oligodendrocyte-specific protein (also: Claudin-11) |
PETZ | Präklinisches Experimentelles Tierzentrum; animal facility of the University Erlangen-Nürnberg |
PGP 9.5 | Protein gene product 9.5 |
PLP / PLP1 | Proteolipid protein 1 (also: lipohilin) |
PNS | Peripheral nervous system |
PSC | Perisynaptic Schwann cell |
RT-PCR | Reverse transcriptase polymerase chain reaction |
TAE | Tris/acetate/EDTA-buffer |
TBS | Tris-buffered saline |
tdT | tdTomato |
α-BT | α-Bungarotoxin |
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N° | Staining Protocol | Antibody Setup |
---|---|---|
① | Quadruple staining of PLP, Calbindin D28k, MBP and Hoechst for Cerebellar Tissue | Primary Antibodies |
Rat anti-PLP Guinea pig anti-Calbindin D28k Rabbit anti-MBP | ||
Secondary Antibodies | ||
Donkey anti-rat Alexa 488 Goat anti-guinea pig Alexa 555 Donkey anti-rabbit Alexa 647 Hoechst | ||
② | Quadruple staining of PLP, Calbindin D28k, GFAP and Hoechst for Cerebellar Tissue | Primary Antibodies |
Rat anti-PLP Guinea pig anti-Calbindin D28k Rabbit anti-GFAP | ||
Secondary Antibodies | ||
Donkey anti-rat Alexa 488 Goat anti-guinea pig Alexa 555 Donkey anti-rabbit Alexa 647 Hoechst | ||
③ | Triple Staining of GFAP, Synaptophysin and α-BT for Skeletal Muscle and Esophagus | Primary Antibodies |
Rabbit anti-GFAP Guinea pig anti-Synaptophysin | ||
Secondary Antibodies and Toxins | ||
Donkey anti-rabbit Alexa 488 Donkey anti-guinea pig Alexa 647 α-Bungarotoxin Alexa 555 | ||
④ | Triple staining of MBP, Synaptophysin and α-BT for Skeletal Muscle | Primary Antibodies |
Rabbit anti-MBP Guinea pig anti-Synaptophysin | ||
Secondary Antibodies and Toxins | ||
Donkey anti-rabbit Alexa 488 Donkey anti-guinea pig Alexa 647 α-Bungarotoxin Alexa 555 | ||
⑤ | Quadruple staining of PLP, Synaptophysin, α-BT and Hoechst for Skeletal Muscle and Esophagus | Primary Antibodies |
Rat anti-PLP Guinea pig anti-Synaptophysin | ||
Secondary Antibodies and Toxins | ||
Donkey anti-rat Alexa 488 Donkey anti-guinea pig Alexa 647 α-Bungarotoxin Alexa 555 Hoechst | ||
⑥ | Triple Staining of ChAT, MBP and α-BT for Skeletal Muscle | Primary Antibodies |
Goat anti-ChAT Rabbit anti-MBP | ||
Secondary Antibodies and Toxins | ||
Donkey anti-goat Alexa 647 Donkey anti-rabbit Alexa 488 α-Bungarotoxin Alexa 555 | ||
⑦ | Triple Staining of ChAT, Synaptophysin and α-BT for Skeletal Muscle | Primary Antibodies |
Goat anti-ChAT Guinea pig anti-Synaptophysin | ||
Secondary Antibodies and Toxins | ||
Donkey anti-goat Alexa 488 Donkey anti-guinea pig Alexa 647 α-Bungarotoxin Alexa 555 | ||
⑧ | Triple Staining of βIII-tubulin, MBP and α-BT for Skeletal Muscle | Primary Antibodies |
Rabbit anti-βIII-tubulin Chicken anti-MBP | ||
Secondary Antibodies and Toxins | ||
Donkey anti-rabbit Alexa 488 Goat anti-chicken Alexa 647 α-Bungarotoxin Alexa 555 |
N° | Staining Protocol | Antibody Setup |
---|---|---|
① | Quadruple Staining of MBP, ChAT, α-BT and Hoechst | Primary Antibodies |
Rabbit anti-MBP Goat anti-ChAT | ||
Secondary Antibodies and Toxins | ||
Donkey anti-rabbit Alexa 488 Donkey anti-Goat Alexa 647 α-Bungarotoxin Alexa 555 Hoechst | ||
② | Triple Staining of DsRed, PLP and Protein Gene Product 9.5 (PGP 9.5) in PLP-CreERT2 x tdT mice | Primary Antibodies |
Rabbit anti-DsRed (This antibody also binds to tdTomato) Rat anti-PLP Guinea pig anti-PGP 9.5 | ||
Secondary Antibodies | ||
Donkey anti-rabbit Alexa 647 Donkey anti-rat Alexa 488 Donkey anti-guinea pig DYE 405 | ||
③ | Triple Staining of GFAP, PGP 9.5 and Hoechst | Primary Antibodies |
Rabbit anti-GFAP Guinea pig anti-PGP 9.5 | ||
Secondary Antibodies and Toxins | ||
Donkey anti-rabbit Alexa 488 Goat anti-guinea pig Alexa 555 Hoechst | ||
④ | Double Staining of GFP and DsRed in GFAP-EGFP x PLP-DsRed1 Mice | Primary Antibodies |
Chicken anti-GFP Rabbit anti-DsRed | ||
Secondary Antibodies and Toxins | ||
Goat anti-chicken Alexa 647 Donkey anti-rabbit DYE 405 | ||
⑤ | Triple staining of βIII-Tubulin, MBP and α-BT | Primary Antibodies |
Rabbit anti- βIII-tubulin Chicken anti-MBP | ||
Secondary Antibodies and Toxins | ||
Donkey anti-rabbit Alexa 488 Donkey anti-chicken Alexa 647 α-Bungarotoxin Alexa 555 |
Gene | Forward Primer Reverse Primer Primer Reference | Cycle Conditions | |||
---|---|---|---|---|---|
β-Actin (154 bp) |
(F) 5′-GGCTGTATTCCCCTCCATCG-3′ (R) 5′-CCAGTTGGTAACAATGCCATGT-3′ Self-Designed | Initial Denaturation | 10 min | 95 °C | |
35 Cycles | Denaturation | 45 s | 95 °C | ||
Annealing | 30 s | 57 °C | |||
Extension | 45 s | 72 °C | |||
Final extension | 10 min | 72 °C | |||
GFAP (199 bp) | (F) 5′-CAACGTTAAGCTAGCCCTGGACAT-3′ (R) 5′-CTCACCATCCCGCATCTCCACAGT-3′ Shi et al. [77] | Initial denaturation | 10 min | 95 °C | |
35 Cycles | Denaturation | 45 s | 95 °C | ||
Annealing | 30 s | 60 °C | |||
Extension | 45 s | 72 °C | |||
Final extension | 10 min | 72 °C | |||
PLP (218 bp) | (F) 5′-AGCGGGTGTGTCATTGTTTGGGAA-3′ (R) 5′-ACCATACATTCTGGCATCAGCGCA-3′ Chew et al. [78] | Initial denaturation | 10 min | 95 °C | |
35 Cycles | Denaturation | 45 s | 95 °C | ||
Annealing | 30 s | 58 °C | |||
Extension | 45 s | 72 °C | |||
Final extension | 10 min | 72 °C | |||
MBP (342–642 bp) | (F) 5′-ATGGCATCACAGAAGAGACC-3′ (R) 5′-CATGGGAGATCCAGAGCGGC-3′ Ye et al. [79] | Initial denaturation | 10 min | 95 °C | |
35 Cycles | Denaturation | 45 s | 95 °C | ||
Annealing | 30 s | 56 °C | |||
Extension | 45 s | 72 °C | |||
Final extension | 10 min | 72 °C | |||
MAG (355–400 bp) | (F) 5′-CTCTATGGCACCCAGAGCCT-3′ (R) 5′-TGTCCTTGGTGGGTCGTTTT-3′ Ye et al. [79] | Initial denaturation | 10 min | 95 °C | |
35 Cycles | Denaturation | 45 s | 95 °C | ||
Annealing | 30 s | 56 °C | |||
Extension | 45 s | 72 °C | |||
Final extension | 10 min | 72 °C | |||
OSP (339 bp) | (F) 5′-GATTGGCATCATCGTCACAACG-3′ (R) 5′-AGCCAGCAGAATAAGGAGCACC-3′ Hellani et al. [80] | Initial denaturation | 10 min | 95 °C | |
35 Cycles | Denaturation | 45 s | 95 °C | ||
Annealing | 30 s | 50 °C | |||
Extension | 45 s | 72 °C | |||
Final extension | 10 min | 72 °C | |||
MOG (841 bp) | (F) 5′-GACCTCAGCTTGGCCTGACCC-3′ (R) 5′-TGCTGGGCTCTCCTTCCGC-3′ Delarasse et al. [81] | Initial denaturation | 5 min | 94 °C | |
35 Cycles | Denaturation | 1 min | 95 °C | ||
Annealing | 1 min | 66 °C | |||
Extension | 3 min | 72 °C | |||
Final extension | 5 min | 72 °C |
Primary Antibodies | Host Species | Dilution | Source (Catalogue Number) |
---|---|---|---|
Calbindin D28k | Guinea pig | 1:100 | Synaptic Systems Göttingen, Germany (214 004) |
ChAT | Goat | 1:40 | Millipore Temecula, CA, USA (AB144P–1ML) |
DsRed | Rabbit | 1:1000 | Takara Bio Mountain View, CA, USA (632496) |
GFAP | Rabbit | 1:800–1:2000 | Dako Glostrup, Denmark (Z0334) |
GFP | Chicken | 1:1000 | ThermoFisher Waltham, MA, USA (A10262) |
MBP | Rabbit | 1:200–1:500 | Abcam Cambridge, UK (ab40390) |
MBP | Chicken | 1:500–1:3000 | Abcam Cambridge, UK (ab134018) |
PGP 9.5 | Guinea pig | 1:500 | Fitzgerald Ind. Acton, MA, USA (20R–PG011) |
PLP (PLP1) | Rat | 1:1000 | Kind gift from Wendy B. Macklin |
Synaptophysin | Guinea pig | 1:1000 | Synaptic Systems Göttingen, Germany (101 004) |
βIII-Tubulin | Rabbit | 1:500–1:4000 | Abcam Cambridge, UK (ab18207) |
Secondary Antibodies and Toxins | Dilution | Source (Catalogue Number) | |
Donkey Anti-Chicken Alexa 647 | 1:1000 | Jackson ImmunoResearch West Grove, PA, USA (703–605–155) | |
Goat Anti-Chicken Alexa 647 | 1:1000 | Jackson ImmunoResearch West Grove, PA, USA (103–605–155) | |
Donkey Anti-Goat Alexa 488 | 1:1000 | Molecular Probes Eugene, OR, USA (A11055) | |
Donkey Anti-Goat Alexa 647 | 1:1000 | Molecular Probes Eugene, OR, USA (A21447) | |
Donkey Anti-Guinea Pig DYE 405 | 1:200 | Jackson ImmunoResearch West Grove, PA, USA (706–475–148) | |
Goat Anti-Guinea Pig Alexa 555 | 1:1000 | Molecular Probes Eugene, OR, USA (A–21435) | |
Donkey Anti-Guinea Pig Alexa 647 | 1:1000 | Jackson ImmunoResearch West Grove, PA, USA (706–605–148) | |
Donkey Anti-Rabbit DYE 405 | 1:200 | Jackson ImmunoResearch West Grove, PA, USA (711–475–152) | |
Donkey Anti-Rabbit Alexa 488 | 1:1000 | ThermoFisher Waltham, MA, USA (A–21206) | |
Donkey Anti-Rabbit Alexa 647 | 1:1000 | Molecular Probes Eugene, OR, USA (A31573) | |
Donkey Anti-Rat Alexa 488 | 1:1000 | ThermoFisher Waltham, MA, USA (A–21208) | |
α-Bungarotoxin Alexa 555 | 1:1000 | Molecular Probes Eugene, OR, USA (B35451) | |
Hoechst | 1:1000 | Sigma-Aldrich St. Louis, MO, USA (H6024) |
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Kapitza, C.; Chunder, R.; Scheller, A.; Given, K.S.; Macklin, W.B.; Enders, M.; Kuerten, S.; Neuhuber, W.L.; Wörl, J. Murine Esophagus Expresses Glial-Derived Central Nervous System Antigens. Int. J. Mol. Sci. 2021, 22, 3233. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22063233
Kapitza C, Chunder R, Scheller A, Given KS, Macklin WB, Enders M, Kuerten S, Neuhuber WL, Wörl J. Murine Esophagus Expresses Glial-Derived Central Nervous System Antigens. International Journal of Molecular Sciences. 2021; 22(6):3233. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22063233
Chicago/Turabian StyleKapitza, Christopher, Rittika Chunder, Anja Scheller, Katherine S. Given, Wendy B. Macklin, Michael Enders, Stefanie Kuerten, Winfried L. Neuhuber, and Jürgen Wörl. 2021. "Murine Esophagus Expresses Glial-Derived Central Nervous System Antigens" International Journal of Molecular Sciences 22, no. 6: 3233. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22063233