Biological Specimen Preparation

BSP Laboratory

Specimen quality is often a deciding factor in acquiring high-quality microscopy images. KGLMF established the Biological Specimen Preparation (BSP) Laboratory to aid researchers in preparing their samples for imaging on KGLMF equipment. BSP offers a range of in-house sample preparation services, equipment access and training for researchers to prepare their own samples as required.

There are 3 main areas in BSP. For more information on any of the following services, please contact Dr. Renee Whan .

1. Routine and Advance Histology

We offer both routine and advanced histology specimen preparation. Our in-house sample preparation services include:

  • Sample processing (Paraffin embedding)
  • Slide sectioning (Paraffin and Frozen)
  • Histology staining
  • Immunohistochemical staining (single/multiple)
  • Special stains
  • Tissue Microarrays (TMA)
  • Multiplexed immunofluorescence staining

We also offer access to specimen preparation equipment access and training for researchers to prepare their own samples as required.

To access this service, please provide the completed and signed request forms CRYO Sectioning, Tissue Processing & Sectioning or Immunolabelling to Fei Shang or Iveta Slapetova

Please click here for the current charges for this service.

2. Slide Scanning

We offer both bright-field (BF) and fluorescence (FL) slide scanning services. We also offer access and training to the slide scanning equipment for researchers to scan their own slides as required. BF slide scanning is performed on the Aperio XT scanner and the images are viewed in the ImageScope software. We can assist you with the setup of the slide analysis if required.

In addition, we provide access to the Vectra Polaris system for both BF and FL slides and multiplexed immunofluorescent stained specimens (OpalTM Kit). The Vectra Polaris can process specimens prepared as whole tissue sections or tissue microarray (TMA) slides.

To access this service, please provide the completed and signed BSP Slide Scanning Request Form to Florence Tomasetig or Iveta Slapetova or Merryn Brettle

Please click here for the current charges for this service.

3. Specimen Processing for Electron Microscopy

We offer services in specimen processing for Electron Microscopy (EM) and Correlative Light and Electron Microscopy (CLEM), in collaboration with the Electron Microscopy Unit (EMU). These services include:

  • Cryo fixation (Leica ICE high-pressure freezing)
  • Chemical specimen fixation (glutaraldehyde/paraformaldehyde)
  • EM sample processing (microwave-assisted EM processing or AFS2-automated freeze substitution)
  • CLEM – Light microscopy

To access this service, please provide the completed and signed BSP CLEM Services Request Form to Iveta Slapetova.

Please click here for the current charges for this service.



BSP Grouping Protocol

Price (Ex. GST)


Price (Ex GST)


Embedding Paraffin Embedding $5.00 $9.00  Per Block
  Paraffin Slide Sealing $1.00 $2.00 Per Slide
Sectioning Paraffin Sectioning $5.00
For first slide from each block
For subsequent slides from same block
  Cryosectioning $5.00
For first slide from each block
For subsequent slides from same block
  Microtome DIY^ $20.00 $36.00 Per Hour (1 hour training required for new users)
  Vibratome DIY^ $20.00 $36.00 Per Hour (1 hour training required for new users)
  Cryomicrotome DIY^ $25.00 $45.00 Per Hour (1 hour training required for new users)
  Microtomy Subscription DIY^ (Yearly) $400.00 N/A

For usage > 20 hours within the calendar year

(1st Jan - 31st Dec)

Staining H&E Staining $5.00
For first slide of each stain
For subsequent slides of same stain
  Immunostaining - Chromogenic DAB $20.00 $36.00 Per one antibody on slide (does not include sectioning; primary antibodies to be provided by user)
  Immunostaining - Fluorescence $20.00 $36.00 Per one antibody on slide (does not include sectioning; primary and secondary antibodies to be provided by user) -  additional primary antibody $20/$35 each
  Specialised Stains TBD TBD Pricing depends on protocol

^     DIY – Do It Yourself (self service)

Please click here for more details about Histology/BSP services.


Immunolabeling is a tool that enables researchers to visualize spatial architecture of tissue sample and cell structures using antibodies targeted against specific proteins. The use of antibodies on fixed tissue and cells allows us to study cell biology, disease pathology, its progress and treatment response on cellular and subcellular level. With the current development in systems biology the demand has increased to not only identify important biological markers but to put them into a spatial context back inside the tissue and cell.

Using conventional chromogenic (IHC) or fluorescence (IF) immunolabeling we can visualize from 1 to 4 markers in one sample. Multiplex immunolabeling addresses the limitation of detection of more than 4 markers and takes us on a discovery road of unlimited marker combinations.

KGlmf is currently working with 3 different multiplex modalities.


  1. Multiplex immunohistochemistry using commercialized methods
  2. Autonomous multiplexing on a microscope with real time processing and analysis
  3. Single molecule multiplexing – DNA paint


1 - Multiplex immunohistochemistry using commercialized methods

There are multiple commercial methods available on the market that enable antibody multiplexing on tissue. The methods are based on use of primary antibody but vary in the use of secondary antibody and their application to the sample. As an example, Ultivue’s InSituPlex technology and Akoya Biosciences’ CODEX technology both rely on oligo-labelled primary antibodies for tissue imaging. The primary antibodies are conjugated with specific DNA oligonucleotide which is either amplified or visualized using fluorescently labeled DNA probe. Both technologies allow for simultaneous labelling of 4-40 antibodies however due to the specific design of the primary antibodies can be costly and only limited amount of antibodies is available on the market..

A more simple approach is to use the primary antibody in sequential labelling process followed by labelling of secondary antibody resulting in specific fluorescent label. An example of this technology is Akoya Biosciences’ Opal Opal labelling relies on HRP-labeled secondary antibodies to catalyze the reaction of tyramide signal amplification. Once the fluorescently labelled tyramide (Opal) has covalently bound to tyrosine residues on and around the protein of interest, heat retrieval is used to remove the antibodies without disturbing the Opal fluorescent signal; the cycle can then be repeated. Any primary antibody can be used for this technology. In KGLMF, the Opal workflow has been optimized for use on the automated IHC stainer Leica BOND RX and is integrated with Phenoptics instrumentation (Vectra Polaris slide scanner and Inform software analysis).

The optimization of the Opal multiplexing is demanding as all OPAL fluorescent intensities must be well balanced to eliminate spectral crosstalk between each antibody and takes between 6-8 weeks.

KGLMF offers OPAL staining of up to 8 antibodies. We carry out this as a service as well as we provide training for processing and analysis. If requested as a service the package includes antibody optimization using DAB IHC, OPAL monoplex and multiplex, final study multiplexing, Imaging on Vectra Polaris and assistance with image analysis.

Pricing list for OPAL services for 2022:


KGLMF OPAL cost for 2021

UNSW and affiliated institutes

Australian universities



$ per slide

$ per slide

$ per slide

BOND reagents




BOND single immunohistochemistry DAB




BOND immunohistochemistry positions DAB








































KGLMF OPAL project fee





$ per hour

Staff engagement per hour




Leica Bond/Vectra Polaris peak hour




Leica Bond/Vectra Polaris off peak hour




OPAL project fee is valid for one calendar year and covers the following:

  • optimization of antibodies using DAB and OPAL by KGLMF staff
  • imaging on Vectra Polaris and other microscopes associated with antibody optimization using DAB and OPAL delivered by KGLMF staff
  • initial image analysis, training and support in image analysis
  • regular reporting

*Prices are subject to changes due to exchange rate fluctuations or price rises by suppliers If you require more information OPAL services provided by KGLMF and on your project price estimate, please contact Iveta Slapetova.

KGLMF has optimised the following antibody panel on human tonsil tissue:

CD3, CD4, CD8, CD11b, CD20, CD103, CD140b, CD68, PD-L1, FoxP3, PD1, PanCK

If you require to use any of these antibodies, please contact us directly.


2 – Autonomous multiplexing on a microscope with real-time processing and analysis

In KGLMF multiplexing can be performed on the epifluorescence or confocal microscope using Centoni microfluidics device with real-time processing and analysis happening at the microscope Nikon Ti2 and offline. This technology uses a system of primary antibodies and secondary antibodies labelled with Alexa Fluor fluorophores. The antibodies are applied to the sample using microfluidics device and resulting fluorescent signal is imaged on the Nikon Ti2 microscope. On completion of the imaging and online analysis the antibodies are eluted, and sample is incubated with new antibody. The whole process is repeated for as many antibodies as desired. The procedure is fully automated and integrated with the Nikon Ti2 microscope via Nikon NIS Elements JOBS module.

KGLMF currently offers a training and support for groups interested in using this technology. Should you wish to obtain more information please contact Michael Carnell.

3 – Single molecule autonomous multiplexing - DNA points accumulation for imaging in nanoscale topography – DNA PAINT

Super-resolution microscopy, such as single-molecule localization microscopy, enables the visualization of biomolecules at the nanoscale. BMIF houses a Zeiss Elyra 7 and a Nikon NSTORM microscope that are used for single-molecule imaging. Although they can both image with a variety of modalities the use of DNA paint lends itself most suitably to multiplexing. Single-molecule localisation historically has been limiting in the number of channels that can be sequentially imaged, due to spectral restrictions and also a reduced selection of suitable dyes. DNA paint has overcome many of these limitations. After using antibodies to label your structure of interest with a DNA oligomer, a fluorescent dye labelled bound to a complementary sequence is added. Bound dye can be distinguished from that which is freely diffusing due to its stable position. After some time, this will unbind and rebind providing an opportunity to temporarily visualise single molecules, gradually building up a super-resolution image. With the use of automated microfluidics this process can be repeated with a new set of freely diffusing DNA oligomers with a complimentary sequence targeted towards a different set of oligo-labelled antibodies, facilitating the ability to image numerous rounds of different structures one after the next, even using the same fluorophore. KGLMF currently offers training and support for groups interested in using this technology.

Should you wish to obtain more information please contact Michael Carnell.