Etchevers:Notebook/Genomics of hNCC/2008/11/07

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Aliquots of antibodies and immunocytochemistry

Aliquots to come

  • The anti-fluorescein antibody from Invitrogen had not arrived yet. Froze the anti-mouse rabbit IgG in its 2 ml aliquot - freeze-thaw is alright I suppose. Use what have on hand for now (a few 3 μL frozen aliquots from Jean-Claude's time).
  • 1 ml of Ki-67 7B11 clone mouse antibody 23 mg/ml, lot 428074A, store at 2-8°C. Zymed (Invitrogen) 18-1092Z. This will not bind in non-human primates according to fact sheet. If 5 μL aliquots will be 200 of them! arrgh. Let's say want to make up 5 mls of 10 µg/ml diluted; this is approximately 1:250. Then you can aliquot by 20 μL which is only 50 of them. On the other hand, perhaps ok to dip repeatedly into stock, make up 10x 100 μL instead. Aliquots of 50 μL in fridge.
  • 111.11 μL of anti-Otx2 pAb at 0.9 mg/ml ab21990-100 lot 553489 Abcam. This was aliquotted by 4 μL (3.6 μg) per tube, kept in freezer. *Heather 16:26, 7 November 2008 (EST):
  • 100 μL of anti-Pax6 pAb at 1 mg/ml ab 5790-100 lot 517447 Abcam (where is this? as of 25/11)
  • 50 μg (50μL at 1mg/mL) of pAb anti-Rax ab23340-50 lot 557008 Abcam. Looking at it on 25-11 to aliquot it, and it seems empty! Am calling Abcam now. They will replace free of charge, but none is in stock, so will have to wait until December 3rd or so. *Heather 07:05, 25 November 2008 (EST):

(I believe there is plenty of anti-Sox2 still at -20°C.)

  • 1 mL mAb to B2M at 0.2 mg/ml = 200 μg, ab19883-200 lot 491585 Abcam. IgG2a. Aliquotted by 100 μL and kept in fridge.
  • 100 μL of anti-beta III tubulin / TuJ1 MAB1637 from Chemicon/Millipore, stored at at -20°C, thawed on ice. Lot LV1462090. IgG1. Aliquotted by 5 μL and kept in freezer
  • Heather 12:44, 25 November 2008 (EST):
  • 100 μL at 1 mg/mL of anti-CNPase MAB326 from Chemicon/Millipore stored and recommended storage in fridge. Lot LV1438362. IgG1. Aliquotted by 10 μL and kept in fridge
  • 1 mL at 0.2 mg/mL of anti-PGD2 synthase (for keratocytes) from Santa Cruz, sc-30067/FL-190, lot J1405. Aliquotted by 50 μL and kept in fridge
  • 250 μL anti-fluorescein-R-phycoerythrin (bright pink!) from Millipore, received 18-11-08. A few aliquots of 25 μL stored in fridge in metal canister, seven are at -20°C as packaging says stable in fridge for 3 months and longer in freezer.

Today's protocol for immunocytochemistry: if I had all the antibodies on hand

  • Cells on coverslips had been fixed in 4% paraformaldehyde, 150 mM sucrose and kept in PBS/150 mM sucrose since.
  • Blocking reagent 1x in 0.1% Tween 20/phosphate-buffered saline/5% SVF from 11:50-13:00 at 4°C and then 2 drops of ImageIT-Fx was applied to each at RT.
  • Primary antibodies were used at a dilution of 1:100 (lazy me) and incubated for 1h from 13:30 at 37°C
  • 4 × 5-min washes PBT.
  • Secondary antibodies conjugated to Alexa Fluor fluorophores were used at a 1:100 dilution and were incubated for 50 minutes at room temperature in the dark.
  • 3 × 5-min washes PBT.
  • Coverslips mounted with ProLong Gold mounting media containing DAPI.

However, I have not received the Millipore antibodies yet.

  • Heather 03:51, 7 November 2008 (EST):

So, I thought - but two out of three Millipore Abs and one Santa Cruz are localized and added above. Called Millipore and they said not foreseen to ship the anti-tyrosinase before mid-December (05-647)! They will try to negotiate to provide me with an expired aliquot. Will see later this afternoon.

Here are coverslips that I dispose of; the ones briefly incubated in Sigma's S3184 Stemline Neural Stem Cell Expansion Media (N) must have anti-TuJ1 on them.

H-T: 3 + 1N H-C: 3 + 1N C6000: 3 + 1N 401: 3 + 1N S: 4 + 1N

Copy combinations later.

  • Heather 10:15, 7 November 2008 (EST):

Here they are: For all five conditions (hESCGro cephalic and trunk, C6000 cephalic, Panexin 400 cephalic and Stempan cephalic cells, two pairs of antibodies:

  • TuJ1 (Alexa Fluor 633) + beta 2 microglobulin (Alexa Fluor 555). This should mark all cells with the latter and a few at least (I hope) with the former, can visualize under the confocal, but will not see the DAPI as would in the histology room.
  • CNPase (glial/melanocyte) (Alexa Fluor 633) + alpha SMA (Alexa Fluor 555). Much as above, but B2M will be diffuse.
  • For the extra Stempan slide, tried out the anti-PGD2 at 1:100, could even go down to 1:50 apparently. Used Alexa Fluor 546 in the end.

Left one coverslip per condition for the potential anti-tyrosinase that Millipore in an access of good salesmanship will provide for me, even if it is nominally expired.

  • Heather 16:44, 7 November 2008 (EST):