ESO wikiadmin: Created page with "{| class="wikitable" |+ !Materials |- |'''Fixation buffer''': 3% Formaldehyde, 2% Glutardialdehyde, 10 mM CaCl₂, 10 mM MgCl₂, 90 mM Sucrose in 100 mM HEPES, pH 7.2 |- |'''Washing Buffer''': 10 mM CaCl₂, 10 mM MgCl₂, 90 mM Sucrose in 100 mM HEPES, pH 7.2 |- |'''HEPES buffer:''' 100 mM, pH 7.2 |- |'''Graded ethanol series:''' 30%, 50%, 70%, 90%, 100% ethanol p.a. |} === Procedure: === '''1A. ''Organs''''' of infected animals are..."

2025-05-20T08:54:19Z

Created page with "{| class="wikitable" |+ !Materials |- |'''Fixation buffer''': 3% Formaldehyde, 2% Glutardialdehyde, 10 mM CaCl2, 10 mM MgCl2, 90 mM Sucrose in 100 mM HEPES, pH 7.2 |- |'''Washing Buffer''': 10 mM CaCl2, 10 mM MgCl2, 90 mM Sucrose in 100 mM HEPES, pH 7.2 |- |'''HEPES buffer:''' 100 mM, pH 7.2 |- |'''Graded ethanol series:''' 30%, 50%, 70%, 90%, 100% ethanol p.a. |} === Procedure: === '''1A. ''Organs''''' of infected animals are..."

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{| class="wikitable"
|+
!Materials
|-
|'''Fixation buffer''': 3% Formaldehyde, 2% Glutardialdehyde, 10 mM CaCl2, 10 mM MgCl2, 90 mM Sucrose in 100 mM HEPES, pH 7.2
|-
|'''Washing Buffer''': 10 mM CaCl2, 10 mM MgCl2, 90 mM Sucrose in 100 mM HEPES, pH 7.2
|-
|'''HEPES buffer:''' 100 mM, pH 7.2
|-
|'''Graded ethanol series:''' 30%, 50%, 70%, 90%, 100% ethanol p.a.
|}

=== Procedure: ===
'''1A. ''Organs''''' of infected animals are excised, washed with PBS, and incubated in cold fixation buffer over night at 4°C. (Time depends on size and texture of tissue)

'''1B. ''Isolated cells''''' grown on glass coverslips are fixed by gentle pipetting of 1 ml of cold fixation buffer directly onto the side of the coverslip, thereby slowly pushing the cell culture medium up, creating a layer of fixative underneath the cell culture medium. Do not mix at this point ! Incubate for 5 min at RT, then aspirate medium and fixative and add 1 ml of fresh fixation buffer for at least 30 min at RT. Samples can be stored before further processing in fixative at 4°C.

''(Note: Used fixation buffer should be collected in a separate waste bottle!)''

'''2.''' Fixed samples are washed 3 times with washing buffer (3 x 10 min)

''Optional: 1% OsO4 in dH2O for 60 min at 4°C''

Samples are washed 3 times in 100 mM HEPES pH 7.2 (3 x 10 min).

'''3. ''' Dehydration in a graded series of ethanol:

30% ethanol 4°C, 7 min

50% ethanol 4°C, 10 min

70% ethanol 4°C, 10 min ''(storage overnight at 4°C is possible at this step)''

80% ethanol RT, 10 min

90% ethanol RT, 10 min 3x100% ethanol RT, 10 min

''(For tissues use 60 min incubation times)''

'''4. ''' After critical point drying from liquid CO2, samples are sputter-coated with 5 nm gold-palladium in a BAL-TEC SCD 030 and examined at 15 kV accelerating voltage in a Zeiss Auriga or Zeiss Evo scanning electron microscope using the secondary electron detector.

After critical point drying from liquid CO2, 8 Zyklen a 10 min, Enddruck: xx bar@ 44°C, samples are sputter-coated with 6 nm Pt and examined at 15 kV accelerating voltage in a Zeiss Auriga or Zeiss Evo scanning electron microscope using the secondary electron detector.

Scanning electron microscopy (SEM) - Revision history