Histologická laboratoř

Přístrojové vybavení (v angličtině)

Equipment

The most important laboratory equipment consists of a set of Leica devices – tissue processor, paraffin embedding station, two microtomes, cryotome and vibratome. There is also a set of trays for tissue deparaffination and H&E stain and pressure cooker for antigen retrieval. Users must be instructed before starting their work on these instruments.

Tissue processor

Tissue processor HistoCore PEGASUS is an automated device for tissue dehydration and wax penetration, with capacity up to 400 histology cassettes. Samples in cassettes are placed in the metal racks in 70% ethanol. They are processed according to the following program. The program runs overnight and lasts 12 hours. Tissue processor is operated by the laboratory staff.

Documents	EN manual, CZ manual
Reservation	reservation form

Paraffin embedding station

Paraffin embedding station (Leica HistoCore Arcadia H) and cold plate (Leica Histocore Arcadia C) are the instruments for creating paraffin blocks, self-operated by registered users. The wax-penetrated tissue samples are placed to the heated drawer by the staff after processing. Users will create paraffin blocks for tissue sectioning according to their individual preferences. Various sizes of the base molds are available.

Documents	HistoCore Arcadia H – EN manual, CZ manual HistoCore Arcadia C – EN manual, CZ manual
Reservation	Calpendo (“Paraffin”)

Microtomes

Microtome Leica RM2255

Two Leica RM2255 is a fully motorized rotary microtome equipped with a smooth-running handwheel.
The RM2255 is primarily designed for motorized sectioning, but is also suitable for manual sectioning applications.
The RM2255 allows the sectioning of samples in four different sectioning modes: single stroke, continuous stroke, step mode and rocking mode. The instrument is equipped with an individually adjustable specimen retraction system, which can be switched on and off by the user. The following accessories are available for any of both microtomes:

carbide knifes
active cooling during sectioning
water bath directly below the knife

Trimming thickness range and increments	1-600 µm
Section thickness range	0.5 – 100 µm
Available feather blade types	A35 – Universal N35 – Hard Tissue R35 – Serial Sections S35 – Routine Sectioning
Documents	EN manual, CZ manual
Reservation	Calpendo (“Microtome 1”, “Microtome 2”)

Vibrating blade microtome Leica VT1200 S

The fully automated Leica VT1200 S vibrating blade microtome is designed for sectioning fixed or unfixed fresh tissue in buffer. Automatic feeding, specimen retraction and the use of a cutting window help to minimize sectioning time. VT1200 S is primarily designed for motorized sectioning, but is also suitable for manual sectioning applications. The vertical deflection of the blade can be measured by the Vibrocheck measurement device. The adjustment of the blade allows minimization of the vertical deflection to below 1 μm, which significantly increases the number of viable cells.

Adjustable amplitude	From 0 – 3 mm, in increments of 0.05 mm
Frequency	Fixed: 85 Hz (± 10 %)
Blade travel speed	0.01 – 1.5 mm/s
Adjustable cutting window	individually programmable front and rear position
Maximum specimen size	33 x 50 x 20 mm
Total vertical specimen stroke	20 mm
Cooling option	Chiller
Return speed	1.0 – 5 mm/s, in increments of 0.5 mm/s
Documents	EN manual, CZ manual
Booking	Calpendo (“Vibratome”)

Cryostat Leica CM1950

The CM1950 is a high-performance cryostat with an encapsulated microtome and separate specimen cooling. It features a UV disinfection system, an integrated extraction system for section waste and an motor for motorized sectioning. The cryostat is designed to produce frozen sections for biological, medical and industrial applications.

Section thickness range	Setting values 1 to 100 µm 1.0 µm – 5.0 µm in 0.5 µm steps 5.0 µm – 20.0 µm in 1.0 µm steps 20.0 µm – 60.0 µm in 5.0 µm steps 60.0 µm – 100.0 µm in 10.0 µm steps
Trimming range	1 – 600 µm (research) 1,0 µm – 10,0 µm in 1,0 µm steps 10,0 µm – 20,0 µm in 2,0 µm steps 20,0 µm – 50,0 µm in 5,0 µm steps 50,0 µm – 100,0 µm in 10,0 µm steps 100,0 µm – 600,0 µm in 50,0 µm steps
Maximum specimen size	50 x 80 mm
Total specimen feed	25 mm
Vertical specimen stroke	59 mm
Specimen retraction	20 µm or off
Specimen orientation	8° (x-, y-axis), 360° rotation of specimen disc
Electric coarse feed	Slow: 300 µm/s, in 20 µm steps Fast: 900 µm/s
Cryochamber	Temperature range: 0°C to -35°C
Specimen cooling	Temperature range: – 10 to – 50 °C
Defrosting of specimen head	manual defrost
Cryochamber defrosting	Automatic cryochamber defrosting: programmable, (hot gas defrost), selectable time, 1 defrost in 24 h or manual hot gas defrost, Defrost time: 12 minutes Automatic shut off defrost: at – 5 °C chamber temperature
Quick-freeze shelf	Minimum temperature: down to – 42 °C, at chamber temp. – 35°C Number of freezing stations: 15 + 2 Defrost: manual hot-gas defrost
Peltier element	Number of freezing stations: 2 Maximum temperature difference: 17 K, at chamber temp. of – 35 °C
Documents	EN manual, CZ manual
Booking	Calpendo (“Cryostat”)

Set of trays for Hematoxylin and Eosin stain

The laboratory is equipped with a fume hood with continuous air extraction, in which containers for deparaffinization and water are placed together with a staining kit for Hematoxylin and Eosin.

Hematoxylin-Eosin staining for paraffin embedded specimens

Deparaffinzation

Xylene
Isopropanol:Xylene
Absolute ethanol
96% ethanol
70% ethanol
dH₂O

Incubate in H&E solution 1 (Hematoxylin)
Rinse in tap water
Differentiate with hydrochloric acid 0.1%
Rinse in flowing tap water (blueing)
Incubate in H&E slution 2 (Eosin)
Rinse in flowing tap water

3×5 min
1x5min
2×5 min
1×3 min
1x3min
1x3min

6 min
10 sec
10 sec
6min
30 sec
30 sec

Dehydratation

dH₂O
96% etnanol
Absolute ethanol
Isopropanol:Xylene
Xylene
Clean (mounting Xylene)

1×3 min
1×3 min
2×5 min
1×5 min
2×5 min
1×5 min

Mount in water free media (solakryl).