We isolate mouse embryos at different stages of gestation from 5.5 dpc. They can be later fixed and stained as a whole or embedded and sectioned.
The HistoPathology (HP) facility aims to combine expertise in histological techniques with scientific input from certified pathologist to provide customers with means for complete analysis of tissues. We offer top quality automatic immunostaining as well as tissue processing, sectioning and most of the common histological stains for both paraffin and cryoblocks.
What is more, we provide a continuously growing list of optimized antibodies that can be used with our staining system. Our service also includes consultation before and in the course of experiment and analysis of results concluded with a pathological report.
We offer an ever-growing list of standardized immunostainings using Leica Bond III automatic stainer. For the list of available antibodies please contact the facility directly.
Set up of a new immunostaining protocol:
For a new marker we offer to set up a staining protocol which will offer optimal staining result. For details please contact the facility directly.
Automatic immunostainer Leica Bond III: recently incorporate in the lab (end of 2011) this is a fully automated immunostainer that allows the performance of immunostainings in a faster and more efficient way, with the same or even better quality comparing with the traditional method. This immunostainer has capacity for 30 slides per run, which would be finished in approximately 3 hours. Since the incorporation of the lab in the CSF our team has improved the quality of the immunohistochemistries (IHC) performed with this instrument (see NEWS section in our web page). Our staff is also working to optimize immunofluorescence (IF) and In-Situ-Hybridization techniques in this automatic immunostainer.
The Leica Bond III automatic immunostainer can be only used by the HistoPathology Lab members. Please remember that researchers are not allowed to work with this instrument. In case you are interested in performing immunostainings with the Leica Bond III please contact with the HistoPathology Lab staff.
In addition, take into account that to avoid the detachment of the tissues during the process:
Tissue dehydrator LOGOS Microwave Hybrid Tissue Processor, Milestone: tissue sample dehydrator acquired for the lab at the end of 2012. This is the instrument that performs the automatic tissue dehydration, clearing and paraffin infiltration. The LOGOS dehydrator competes in quality, versatility and safety with other dehydrators in the market. It can perform 210 tissue blocks per run, with any kind of tissue up to 6 mm thickness.
The LOGOS tissue dehydrator can be only used by the HistoPathology Lab members. Please remember that researchers are not allowed to work with this instrument. In case you have samples to dehydrate, please see “Tissue dehydration protocol”. Also remember that small mesh cassettes or inserts as sponges or thick paper MUST NOT be used, as they interfere with the appropriate dehydration of the samples.
Tissue embedding console system Tissue-Tek TEC, Sakura: this is the instrument to perform the last step to prepare the solid paraffin blocks with the tissues that have been previously dehydrated and embedded in paraffin with the dehydrator instrument. It consists of two modules, the warm paraffin bath and plates, and the cooling plate, both at the same height. This allows that the specimens can be transferred easily from one side to the other in an ergonomic way. The temperature of the paraffin and plates and the cooling plate is programmed at 62˚C and -5˚C, respectively. The cooling plate has capacity for 60 cassettes.
See “tissue embedding protocol” in the PROTOCOLS site in our web page.
Glass coverslip machine, Tissue-TEK GLC-Sakura: this instrument allows the automatic and fast coverslipping of your cell/tissue slides with xylene-based mounting medium. A slide rack, with capacity for 20 slides, will be ready in approximately 20 minutes.
See “Coverslipping with xylene-based mounting medium protocol” in the PROTOCOLS site in our web page.
Slide stainer Microm HMS 740-Thermo Scientific: an innovative slide stainer designed for productivity and safety. It is mainly used to perform automatic “hematoxyline and eosin” staining (HE, the basic staining protocol for the microscopic evaluation of tissues). This slide stainer includes one entry and three exit stations, and has a simultaneous multiprogram feature, allowing the staining of four different slide-racks (30 slides/rack) at the same time.
See “HE staining protocol” in the PROTOCOLS site in our web page.
Cassette printer IDent™-Sakura: the Cassette Printer IDent™ can print up to 2 lines of 16 positions each, with small or large characters. The printer is capable of printing all numbers, symbols and special characters, such as µ, a, ε, Ω, π , ß and roman numbers. The ink is resistant to all reagents commonly used in histology or cytology laboratories.
Slide printer IDent™-Sakura: the Slide Printer IDent™ can print up to 4 lines of 16 positions each, with small or large characters. The printer is capable of printing all numbers, symbols and special characters, such as µ, a, ε, Ω, π , ß and roman numbers. The ink is resistant to all reagents commonly used in histology or cytology laboratories.
See “Cassette printer protocol” and “Slide printer protocol” in the PROTOCOLS site in our web page.
Rotary microtomes, (two MICROM HM 355 and two MICROM HM 355S), Thermo Fisher: the rotary microtomes HM 355 and 355S are universal heavy duty microtomes following ergonomical standards concerning operation and comfort. They can be used for paraffin sectioning, offering a section thickness range from 0.5µm to 100 µm. The sections can then be transferred onto a glass slide and used for a wide variety of staining protocols (basic stainings, immunohistochemistry, in-situ-hybridization or immunofluorescence.
See “Rotatory microtome sectioning protocol” in the PROTOCOLS site in our web page.
Cryostat, MICROM HM 560, Thermo Fisher: microtome to perform frozen or cryoembedded tissues. The structure of the tissues is generally less preserved in a cryosection than in a paraffin section. On the other hand, many antigens which may be degenerated during the paraffin embedding process are kept intact in the frozen tissues, allowing them to be still detectable with an immunostainig. Open top cryostat with ergonomic design for working while standing or sitting. This cryostat offers a section thickness range from 0.5 µm to 100µm. The temperature for the specimen clumper is controlled and ranges from 10°C to -35°C. There is a separate temperature control for the knife carrier down to -35°C. The instrument counts with an automatic approach system (ACA) for quick, precise and safe movement of the specimen towards the blade. We have put a thermometer in the cryostat to check the real temperature in the chamber.
See “Cryostate sectioning protocol” in the PROTOCOLS site in our web page.
Sliding microtome MICROM HM 430, with an adapted fast freezing unit: high precise and ergonomic microtome. The roller-guided knife sledge guidance results in an unachieved smooth running of the knife sledge movement for comfortable and non-tiring working. The section thickness feed that the instrument offers is: from 0 to 10 μm in 1 μm-increments, up to 20 μm in 2 μm-increments and up to 60 μm in 5 μm-increments.
See “Sliding microtome sectioning protocol” in the PROTOCOLS site in our web page.
Vibratome, Leica VT1000S: high quality sectioning equipment for fresh or fixed tissue submerged in water or buffer bath. While used with aeration and a proper submerge buffer may be used for cutting tissue that is still alive. The cut is done by means of vibrating razor blade. The instrument counts with and adjustable 2x large-field magnifier. Sectioning thickness setting from 1 μm to 999 μm, in 1 μm in increase. To quickly accommodate different application needs, a user can alter the frequency between 0 and 100Hz and select the appropriate amplitude between 0.2 to 1mm in 5mm steps. An adjustable specimen retraction feature protects the specimen from damage during the return stroke of the knife.
See “Vibratome sectioning protocol” in the PROTOCOLS site in our web page.
Microscope Axioskop 2 plus Zeiss with a multi-viewing system of 2 additional positions: this microscope has been designed as a universally applicable microscope for applications in biology and medicine, e.g. for the examination of blood and tissues. It counts with five magnifications:
The multi-viewing system allows three users simultaneously view the same specimen: one person in the middle moving the mechanical stage and the focusing drives, and two more persons on the left and right sides. This system features a realistic impression of working with a microscope for all users. An arrow-shaped light pointer, located inside the eyepiece of the microscope, allows the person in the middle to point out relevant structures or findings.
Ovens (Heraeus Instruments and P-Selecta): these ovens are available to users to dry paraffin sections. They are programmed at 50°C-55°C. In each oven there is a thermometer to check the real temperature inside.
Please, do not modify the temperature of the oven without notifying it to the HistoPathology staff.
Cytospin 4-Shandon Thermo Electron Corporation: ultimate thin layer cell preparation system, to centrifugate cell suspensions onto glass slides. It can process 12 specimens at the same time. Using centrifugal principles, the cytocentrifuge deposits cells onto a clearly defined area of a glass slide and allows for the absorption of the residual fluid into the sample chamber's filter card. Cytocentrifugation also constructively flattens cells for excellent nuclear presentation. During operation, the instrument's spinning action tilts the Shandon Cytofunnels upright and centrifuges cells onto the deposition area of the slide, giving all cell types equal opportunity for presentation. In the Load or Stop position, this unique tilting feature reduces cell loss by preventing residual fluid from coming into contact with the prepared slide.
Shandon Cytofunnels are available for IMP/IMBA/GMI users at the HistoPathology Lab.
Animal blood counter, Scil Vet-abc: the Scil Vet-abc is simple to operate and produces a complete three part differential blood count in only 90 seconds. The machine accepts only EDTA blood, and 12 µl of EDTA whole blood is enough for the measurement. No sample preparation is necessary. The results are automatically printed in the printer next to the instrument. Before putting the blood sample in the machine the smart card should be inserted in the instrument. The smart card provides the device with the settings of the animal species corresponding to the sample. There are four smart cards available in the HistoPathology Lab: mouse, rat and rabbit. The fourth smart card is the control card, to be used with an artificial blood sample to assess the instrument.
The parameters measured by the instrument are: Leucocytes, Erythrocytes, Haemoglobin, Haematocrit, RDW, MCV, MCH, MCHC, MPV, Thrombocytes, Lymphocytes %, Monocytes % and Granulocytes %. In addition, three histograms for Leucocytes, Erythrocytes and Thrombocytes.
See “Scil Vet-abc animal blood counter protocol” in the PROTOCOLS site in our web page.
Although the Scil Vet-abc animal blood counter, the scil Reflovet Plus clinical chemistry analyzer and the cobas c 111 analyzer are located in the HistoPathology Lab room, our lab members do not have expertise in these machines. The HistoPathology staff only takes care of the maintenance of these instruments and can explain to users how the Scil Vet-abc animal blood counter and the scil Reflovet Plus clinical chemistry analyzer works to analyze ones sample.
Scil Reflovet Plus, Roche: the scil Reflovet Plus clinical chemistry analyzer provides accurate and reliable results in only 2-3 minutes. Results are expedited thanks to the flexibility of the sample required - whole blood, serum or plasma all work so there is no centrifugation needed. Only 32 µl of sample is needed for the measurement.
See the chemistry values evaluated by scil Reflovet Plus here.
The scil Reflovet Plus is ideal to perform glucose curves or to check liver or renal disease.
See “Scil Reflovet Plus clinical chemistry analyzer protocol” in the PROTOCOLS site in our web page.
Cobas c 111 analyzer, Roche: the cobas c 111 analyzer offers a compact desktop solution to clinical chemistry and electrolyte testing. The machine accepts serum, plasma and urine. The minimum sample volume is: primary tubes-500 µl, sample cup and micro cup-40 µl.
See the list of chemistry values evaluated by the Cobas c111 here.
In order to use the Cobas c 111 the researcher must receive training directly from the technical service of the machine. Please contact the HistoPathology staff for more details.
Although the Scil Vet-abc animal blood counter, the scil Reflovet Plus clinical chemistry analyzer and the cobas c 111 analyzer are located in the HistoPathology Lab room, our lab members do not have expertise in these machines. The HistoPathology staff only takes care of the maintenance of these instruments and can explain to users how the Scil Vet-abc animal blood counter and the scil Reflovet Plus clinical chemistry analyzer works to analyze one sample.
When starting a new project related with histology/pathology please make an appointment with our staff members as early as possible to discuss how to obtain the required results, how the facility can support you, and how to plan a time schedule. Users are encouraged to bring some initial information with them, such as papers that show results similar to those the user would like to obtain or protocols they would like to use.
IMP, IMBA and GMI researchers can use almost all instrumentation in the HistoPathology Lab by themselves, but only once they have been trained by our lab staff (“Infrastructure Usage”, see below).
In addition, the HistoPathology lab provides fee-for-service to both Campus Vienna BioCenter (academic institutions and companies) and external institutions and private companies.
For serviced projects, users on the Campus Vienna BioCenter have priority. Beside that, the projects will be performed in the order they are requested: users turning their requests first will be served first.
The list of available services and prices can be found at the SERVICES and PRICES sites in our web page. In case one service is desired, the user must contact the HistoPathology staff to schedule an appointment (“Starting New Projects”, see above). When agreed, a quotation formular with the list of services required and the final total price will be send to the user. It needs to be confirmed and signed by the user’s group leader, and send back to the HistoPathology Lab member in charge in order to allow the service to begin.
Only trained personnel is allowed to work in the HistoPathology Lab. An introduction to the lab with some safety regulations and the basic training for the usage of the required instrument is provided on an individual basis by the HistoPathology staff at any time of the year. Users must not train new users! To schedule a training slot please contact Agnieszka Piszczek (email@example.com), our histologist in the lab, as early as possible. Remember that waiting times might occur. The duration of the training depends on the necessities of each user, taking approximately one hour. The HistoPathology staff will inform the user when they are ready to use equipment on their own. In case an instrument has not been used for long time, the user should contact ask for an update on usage and, if required, retraining.
If training is not successful or if the user does not comply to the rules of the facility, in particular safety rules, our members will document those issues and contact the user and his/her supervisor. If there is not an improvement after several warnings the head of the HistoPathology Lab can temporarily take action and, after consultation with the user committee, decide about subsequent actions.
Remember that two of the instruments in the lab, the LOGOS dehydration machine and the Leica Bond immunostainer, can ONLY be used by the lab members. Users are not allowed to work with these instruments. The protocol for sample dehydration can be found at the PROTOCOLS site in our web page. In case you are interested in immunostaining please contact our staff members.
Basic safety regulations for the proper work in the lab will be explained to new users in the individual training required to have the permission to work in the lab. General rules for biosafety and lab safety are available in a separate document provided by the IMBA safety officer.
Trained users have access to the HistoPathology Lab 24 hours a day, 7 days a week. The magnetic individual card is required to entry into the lab.
Routine reagents and consumables for histology, as slides, blades etc., are provided by the HistoPathology Lab. Remember to use the resources wisely. Reagents and consumables specific to individual experiments have to be provided by the users at their own cost, e.g., primary antibodies (mainly used secondary antibodies are also provided by the HistoPathology Lab).
Remember that the OCT available at the HistoPathology Lab is for usage in the cryostats in the lab. OCT bottles from the HistoPathology Lab should not be taken out of the lab (e.g., should not be taken to the animal house or the user´s own laboratories).
When available, HistoPathology Lab trained users can be provided with bench space in the lab to run their experiments on site. Users must contact our staff members in order to have a bench space assigned. After the user has finished his/her daily work the bench have to be left clean and orderly. If any equipment is missing, this should be reported to our staff.
Available instruments in the lab must be used with utmost care. Users are encoraged to check back with the facility staff members in case of any doubt, and in case any problem with the instrument occurs. The intructions for the instrument usage available at the PROTOCOLS site in our web page should be followed accurately.
After the usage, the user must clean the instrument and also the area where the instrument is located and could get dirty during the usage.
2015 Publications coming soon ...