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Specimens handling


PREPARATION AND STORAGE OF SPECIMENS:

SPECIMENS:

  1. Frozen sections on adhesive-coated glass slides
  2. Cell smears and cytocentrifuged cells (see procedure for the preparation of cytospins)
  3. Fixative-fixed (Formalin, BouinÝs or B5), paraffin-embedded sections on adhesive-coated glass slides.
SAFETY NOTE: unfixed slides, cytospins, smears of human origin are to be considered potentially infectious. Therefore universal precautions in handling are mandatory.

FROZEN SECTIONS / SMEARS / CYTOCENTRIFUGED CELLS

Frozen sections / smears / cytospins are collected preferably, but not necessarily, on adhesive-coated slides (Fisher Superfrost Plus Cat. 12-550-15), and processed as follows within the next 48 hours:

1- air dry in a cool, dry place (AC) for 1hr up to 48 hr.
2- fix in absolute acetone (new aliquot) for 10 min at RT.
3- dry thoroughly for 15 min.
4- store at RT in a dry cool place (NOT the refrigerator, nor the freezer) for up to total of 48 hrs, from the time of preparation.
5- freeze.


To perform In Situ Hybridization:
PRECAUTIONS:  RNA is very labile in the presence of RNAse, an ubiquitous enzyme present on skin, dirty surfaces etc. The use of clean, untouched glassware, of reagents reserved ad hoc, of DPC-treated water solutions and the use of gloves allows handling the specimen with preservation of cellular RNA.
Frozen sections / smears / cytospins are collected on RNAse-free adhesive-coated slides (Fisher Superfrost Plus Cat. 12-550-15), and processed as follows immediately:

1- air dry in a cool, dry place (AC) for 10 min.
2- fix in buffered formalin for 10 min at RT.
3- wash briefly in sterile PBS and dry for 15 min. at RT.
4- store preferably at -80 o C wrapped in clean Saran Wrap (see below).
Note: this protocol is not suitable for Laser Capture Microdissection..


To proceed with the immunostaining:

1- fix in 10% buffered formalin for 10 min at RT (NOTE rare antigens do not survive formalin fixation: proceed with the immunostains after acteone fixation).
2- wash thoroughly in PBS 3x
3- proceed with the immunostains without drying.
To store the frozen sections (see also here):
pair each slide with another glass slide (with or without section on) facing each other, with a short piece of thread in between. (NOTE you may put up to 5 pairs of slides in the same packet).
wrap in Saran Wrap tightly, so the slides are sealed.
label the slides legibly with a permanent  marker
store at -20 o C (or better at -80 o C)
before  open up the packet, allow the slides to reach room temperature.


FIXED, EMBEDDED SECTIONS (AKA Paraffin sections)

<4 Ám sections are collected on adhesive-coated slides  (Fisher Superfrost Plus Cat. 12-550-15), placed vertically in order to facilitate adhesion, and dried for up to 18 hours.
In order to enhance adhesion, the usual recommendation is to "bake" the slides (30 min at 60 o C or overnight (ON) at 37 o C.)
We found this procedure unnecessary if the slides are prepared correctly. When slides are not dried in vertical position or the water is dirty, or there are air bubbles, baking does not helps at all. Anyway, do not exceed 60 o C and/or 30 min. for drying. Do not use microwave oven for drying.
The slides are stored in dry boxes, at RT for short term storage.