Patience is a virtue

Experimental protocol for immunostaining of patterned cells

1. Prepare patterned coverslips, painstakingly, one at a time, over the course of three days
2. Plate cells onto these coverslips
3. Lovingly feed and tend to the cells for a week
4. On the first day of the experiment, fix, permeabilize, block, and label the cells with primary antibody; place in the refrigerator overnight
5. On the second day, permeabilize and label the cells with secondary antibody
6. Place a drop of gel mount onto a microscope slide in preparation of mounting the coverslips
7. Get tweezers out of the box where you keep the tweezers for the express purpose of picking the coverslip out of the Petri dish
8. Discover tweezers are missing again
9. Spend ten to twenty minutes finding the tweezers
10. Swear about this a little bit
11. Put gel mount onto fresh microscope slides to replace the gel mount that has dried on the first set of slides
12. Painstakingly, and with much frustration, pick the coverslips up out of the Petri dish with tweezers
13. Place them carefully on the gel mount
14. While the gel mount is drying, check the set up of the microscope
15. Discover that both lamps are misaligned again
16. Spend ten to twenty minutes realigning the lamps
17. Swear about this a little bit
18. Bring the supposedly dried and mounted samples over to the microscope, place upside down on the 60x oil objective
19. Adjust the stage to locate a cell in the field of view
20. Discover the gel mount was not actually dry, and your sample has been smeared across the slide and is ruined
21. Swear about this quite a lot