| Date | Laborator | Type of Experiment and important details | Labeling Results (K CPM) | Phosphor-I. results | ISH dipping results |
| 1999 08 19 | EED+? | 35S labelling; Qiagen-purification; Beta-rad CPM count (KK's machine) | GAP43: 123 ;GAPDH:12 ;ChAT: 103. | ||
| 1999 08 19 | EED+? | 35S labelling; Qiagen-purification; Beta-rad CPM count (KF's machine) | GAP43: 230 ;GAPDH:22 ;ChAT: 204. | ||
| 2000 01 05 | EED | 35S labelling; Qiagen-purification; Beta-rad CPM count (KF's machine) | VAChT1: 227; VAChT2: 104; ChAT: 180. | - | - |
| 2000 01 05 | EED | Relabeling: 35S labelling; Qiagen-purification; Beta-rad CPM count (KF's machine) | VAChT1: 295; VAChT2: 226; ChAT: 209. | OK | |
| 2000 03 22 | EED | 35S labelling; Qiagen-purification; Beta-rad CPM count (KF's machine) | VAChT1: 990; ChAT TH: 440; ChAT 136(=TH) 493; ChAT 137: 150. | ||
| 2002 01 07 | EED | Retzius lab: first run. 35S labelling; Qiagen-purification; Beta-rad CPM count (KF's machine) | CGRP: 75; ChAT(#137): 199; FGFR1: 154; IGF-1R: 142; gp130: 149; CNTFRa: 172 | -see purification comparison ChAT/CGRP below | |
| 2002 01 14 | EED | Relabelling: 35S labelling; Qiagen-purification; Beta-rad CPM count (KF's machine) | CGRP: 16; ChAT(#137): 31; FGFR1: 21; IGF-1R: 28; gp130: 76; CNTFRa: 31 | ||
| 2002 01 14 | EED | Test: 35S labelling; Amersham-PharmaciaG50-purification; Beta-rad CPM count (KF's machine) | CGRP-Q: 16 ;CGRP-AP: 98 ; ChAT(#137)-Q: 33; ChAT(#137)-AP: 559 | CGRP/ChAT: AP comp with Q from 020107: same distr; high background w AP. CGRP-Q=best signal/noise | 10-day testbox-no signal. |
| 2002 01 21? | EED | HOT RUN. Hybridisation with probes labelled 20020107. D-series L5 SC. | see above | Skipped.. | 3-wks testbox -no signal.
2-mo (-020402): labelling! Results to use (density slicing) in IGF-1 paper. CGRP upregulation saturated in aged! |
| Next experiment will be with new protocol (alternate ISH-protocol) for 33P and photofilm | Switching to FILM | ||||
| Date | Laborator | Type of Experiment and important details | Labeling Results (K CPM) | Film-results | ISH results |
| 20020319 | EED/EVI | Test. Repeat experiment of 020107 with new purification
(Amersham-Pharmacia ) and 33P isotope.
Tissue: SC thoracic leftovers |
CGRP: 251; ChAT(#137): 1104; FGFR1: 1119; IGF-1R: 631; gp130: 605; CNTFRa: 934 | Friday to Monday (3d): (020322-25)
all except CGRP and ChAT visible labeling of grey matter. No obvious specific localisation to e.g. ventral horn! (Note wrong photofilm -not for 33P) Thursday to Tuesday (5d): (020328-0402) FGFR1-nice motor nucleus labelling. gp130-ok. CGRP-possibly specific labelling (precipitates?). ChAT-very weak signal. CNTFRa weak but anterior horn located signal. IGFR1 -no specific signal! |
Dipped 020408. Developed 020603. Background low. Labelling weak in most cases. Signals seem specific. Isotope probably too decayed at time of dipping. |
| 02040? | EED/BUF | Note: 1. Chat #137=old ChAT, found to be sense and not antisense!2.ChAT #136=new Chat, found to be located to intron and not exon!! | |||
| 020514 | EED | New oligos ordered from MWG (see 02040? above): ChAT, CGRPa and GAP43 (ref: HAJ thesis). Diluted from dry product to 4µg/µl. Stock solution (400ng/µl) prepared from this. | |||
| 20020523 | EED/EVI | Hot run. 33P IGFR-1, CNTFRa, gp130, FGFR1 same as before; CGRPa, ChAT (now "old" but antisense), GAP43 and CGRP reordered;. EVIs sectioning: SCL -D-series; low and high symptoms. Pharmacia purification. | 020523. CNTFRa: 753; FGFR1: 781; IGFR1: 889; gp130: 885; CGRP: 777; ChAT: 898; GAP43: 847. | 020524-020227 (3d). Excellent: All markers but ChAT show specific patterns of labelling. Gap43 overexposed! Unfortunately film has not been evenly held to slide surfaces. Film has been flexed due to lack of support at edges in the case which was not full. Always use additional slides for supporting the film evenly! IGF-1R, ChAT and CNTF placed on film again (020527) to be developed 020603 (7d): clearly specific MN signal absent. | First dipping 020528.Planned test slide dipping times and results:GAP43: 020531(3d), Very nice MN labelling-Ag up almost to sat; CGRP: 020603 (6d), very nice labelling +dynamics; FGFR1: 020607(10), reaching saturation -some dynamics may be lost; strong MN labelling and more; gp130: 020611(14d), very nice dynamics -up in aged! Second dipping 020603, develop testslides 020617 (14d). Testlift performed 020614 -weak signals. New testlift done 020626: CNTFRa saturated, IGFR1 some signal; ChAT? |
| 020529 | EED/EVI | Hot run. Same probes as above (labelled 020523) run on soleus muscle (transverse sections): D1, 3, 11, 28 and yad A1-3. | see above (note time delay...) | 020531-020603(3d). Difficult to evaluate. GAP43 very strong signal; all other weak signals. No clear patterns of labelling. | Dipping(020603): GAP43 testlift 020607(4d); results: ok. counterstaining reveals massive infiltration of nuclei in aged muscle (fibroblasts?)! Specificity of labelling hard to judge. -All other testlift 020618(2w, 1d) -not done on time; lift performed 020626: results hard to judge -high background -probably specific signal (i.e. e.g. located to nuclei for IGFR1) |
| Date | Laborator | Type of Experiment and important details | Labeling Results (K CPM) | Film-results | ISH results |
| 021202 | Ming | New oligos ordered from MWG (totally 12st): Diluted from dry product to 4µg/µl. Stock solution (400ng/µl) prepared from this. | |||
| 021206 | Ming/EVI | 35S labelling; Amersham-PharmaciaG50-purification;
including 18 probes; Final volume of dilution is 100ul, due to some hybridizations
of double or triple probes per slide; Beta-rad CPM count (KF's machine)
1 probe: 1ul labelled probe+l ul ddH2O/101ul HS 2 probes: 1ul probe1+1 ul probe2/101 ul HS 3 probes:1ul probe1+1ul probe2+1ul probe3/101ulHS |
LIFR#167: 643; B2-MG#169: 611; IFNrR#174: 486; B2-MG#170: 391; GFRa-2#124: 231; GDNF#163: 254; LIFR#168: 245; MHC I#171: 164; MHC I#172: 152; Ret-ic#31: 160; GFRa-1#29: 143; GFRa-1#164: 143; GFRa-2#123: 156; GFRa-2#165: 145; GDNF#27: 132; Ret-ic#166: 150; Ret-ic#30: 96; IFNrR#173: 128; | ||
| 021208 | Ming | Hot run. The probes as above (labelled 021208) run on Cervical 7 of SC : B1-B5 and hindbrain: B2+B3; 4 slides per probe or probe combination. | The labelled probes are divided into 3 groups, based
on the CPM values:
GroupI: B2-MG #169+#170; B2-MG #169; B2-MG#170; LIFR #167+#168; LIFR#167; IFNrR#174; IFNrR#173+#174; GroupII: GFRaR-2#124; GFRaR-2#123+#124+#165; GDNF#163; GDNF#27+#163; LIFR#168; MHC I#171; MHC I#172; MHC I#171+#172; Ret-ic#31; GroupIII: GDNF#27; GFRa-1#164; GFRa-1#29; GFRa-1#164+#29; GFRa-2#123; GFRa-2#165; Ret-ic#166; Ret-ec#30; IFNrR#173; |
GroupI 021209-021216 (7d) and 021209-021220
(11d):LIFR#167; LIFR #167+#168; B2-MG#170; B2-MG #169+#170;strong and clear
signals, and single labeling is better than double labeling. For IFNrR#174;
IFNrR#173+#174;no clear signals. GroupII 021209-021220(11d) and 021209-030103(25d): MHC I#171+#172, MHC I#171,LIFR#168, strong and clear signals; but GDNF#163; GDNF#27+#163; GFRaR-2#124; GFRaR-2#123+#124+#165, too weak signals. GroupIII 021209-021220 (11d) and 021209-030103 (25d):GFRa-1#164;GFRa-1#164+#29;Ret-ic#166, weak but clear signals; IFNrR#173; GDNF#27; GFRa-1#29, no clear signals |
Dipped (021219) for 2, 3, 4, 6, 8, 9 weeks based on the film results, the probes showing clear signals include GFRa-1#164+#29, GFRaR-2#123+#124+#165, Ret-ic#166, MHC I#171, IFNrR#173+#174, B2-MG#170, LIFR#167, which will be used in the real experiments |
| 021212 | EED | Labelling protocol test | Standard protocol, half volume protocol and half isotope protocol labelling comparison. LIFR#167 used for testing purposes. Old isotope! | ||
| 030104 | Ming | 35S labelling gp130#159 and IFNrR#175, the same protocol as 021206 | gp130#159: 174; IFNrR#175: 352 | ||
| 030106 | Ming | Real experiment The selected probes (labelled 021208 or 030104) are: GFRa-1#164+#29, GFRaR-2#123+#124+#165, Ret-ic#166, MHC I#171, IFNrR#173+#174, B2-MG#170, LIFR#167, , gp130#159, IFNrR#175, run on Lumber 2 of SC, and EM, FB, HB (K1-K5); Lumber 2 of SC, and EM, FB, MES, UB (3:19, 21, 32, 25, 28), 4 or 5 slides per probe or probe combination | see above (note time delay...) | film exposure: GFRa-1#164+#29 (16d), GFRaR-2#123+#124+#165 (16d), signals are clear; Ret-ic#166 (16d), not so clear signals; MHC I#171 (7d), too strong signals; IFNrR#173+#174, not clear signals; B2-MG#170 (14d), LIFR#167 (17d) , not as clear signals as the testing step; gp130#159 (16d), IFNrR#175(16d), not clear signals | Dipped (030122) for 2.5, 6, 6.5, 8 weeks based on the film results. For gp130#159, IFNrR#175, dipped testing boxes for 40 days and real dipping for 51 days. The probes showing clear signals are: GFRa-1#164+#29, GFRaR-2#123+#124+#165, MHC I#171 |