Dec 06, 2024
Fixation of human kidney biopsies for ultrastructural and molecular preservation for clinical research
- Alana Burrell1,
- Lucy Collinson1,
- Candice Roufosse2
- 1Francis Crick Institute;
- 2Imperial College
Protocol Citation: Alana Burrell, Lucy Collinson, Candice Roufosse 2024. Fixation of human kidney biopsies for ultrastructural and molecular preservation for clinical research . protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzxn8lzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 26, 2024
Last Modified: December 06, 2024
Protocol Integer ID: 104161
Keywords: Fixation, Formaldehyde, Human, Tissue, Kidney, Renal, Biopsy, Needle core, Ultrastructure, Molecular, Light microscopy, X-ray microscopy, Electron Microscopy, Correlative, Multimodal, Clinic
Funders Acknowledgements:
UKRI Medical Research Council
Grant ID: MR/W031426/1
Francis Crick Institute (Wellcome, MRC, CRUK)
Grant ID: CC1076
NIHR Imperial Biomedical Research Centre
Grant ID: NA
Disclaimer
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind.
Disclaimer statement source dx.doi.org/10.17504/protocols.io.bh9nj95e
Abstract
This protocol details the fixation of 1 mm diameter human kidney needle core biopsies in the clinic, compatible with ultrastructural and molecular preservation for downstream clinical research applications, including those that use high resolution imaging with light, X-ray and electron microscopes.
Guidelines
Health & Safety requirements:
Ensure you are familiar with all relevant local Health and Safety documents and procedures (Material Safety Data Sheets, Procedural Risk Assessments, Standard Operating Procedures etc.) before starting.
Procedural Risk Assessments: Handling of formaldehyde fixative
Abbreviations:
- PB: Phosphate buffer
- FA: Formaldehyde
Materials
Materials:
- Fridge, 4ºC
- Balance and weighing dishes
- pH meter
- Serological pipette controller
- 25 ml serological pipettes (sterile)
- 5 ml serological pipettes (sterile)
- Magnetic stirrer plate
- Magnetic stirring bars (for stirring buffers)
- Measuring cylinders (for PB buffer prep)
- 500 mL glass beakers (for PB buffer prep)
- Nalgene rapid-flow sterile disposable filter units with nylon membrane, 500 mL, 0.2 µm pore Thermo Fisher Catalog #151-4020
- 5 ml amber screw cap centrifuge tubes (sterile) Eppendorf‱ Catalog #0030122330
- 30 mL polypropylene Universal Containers (sterile) Sterilin‱ Thermo Fisher Catalog #128CP
- Parafilm Agar Scientific Catalog #AGG3398
Chemicals:
- ddH2O; double distilled water (Sterile). Deionised ultra-pure water can be used in place of ddH2O.
- Na2HPO4; di-Sodium hydrogen orthophosphate, anhydrous VWR (Avantor‱)Catalog #102494C OR Na2HPO4•2H2O; di-Sodium hydrogen orthophosphate dihydrate VWR (Avantor‱)Catalog #28029.260
- NaH2PO4•H2O; Sodium dihydrogen orthophosphate monohydrate VWR (Avantor‱)Catalog #102454R
OR
NaH2PO4•2H2O; Sodium dihydrogen orthophosphate dihydrate VWR (Avantor‱)Catalog #28015.261
4. Formaldehyde 36% (aq) stock TAAB Catalog #F003
Safety warnings
Safety critical: All procedures designated safety critical must be performed in a fume hood and the following personal protective equipment (PPE) must be worn - lab coat, gloves, and eye protection.
Ethics statement
Prior ethics approval should be obtained before working with human tissue according to local rules.
Procedure: Immersion fixation of human tissue
Procedure: Immersion fixation of human tissue
Note
- Human biopsies are usually fixed in formalin (that may contain methanol) and embedded in paraffin, or snap frozen. Both processes destroy the ultrastructure of the sample and are unsuitable for preserving samples that will later be imaged by high resolution light, X-ray or electron microscopy for clinical research.
- In some cases, human biopsies are fixed using glutaraldehyde, or a mix of (para)formaldehyde and glutaraldehyde, for diagnostic electron microscopy. Glutaraldehyde can destroy or mask antigens, leading to issues with downstream immunolabelling for clinical research.
- This protocol was developed to preserve both ultrastructure and molecular antigenicity of human biopsies for downstream clinical research using high resolution imaging modalities.
- The protocol was developed for 1 mm diameter human kidney needle core biopsies. Appropriate solution volumes are indicated.
- For larger human biopsies/tissue, it is recommended that the sample is dissected so that it is thinner than 1 mm in at least one dimension to allow good penetration of the fixative.
- Samples must not dry out during fixation and storage, as this will destroy the ultrastructure.
Prepare buffer and fixative aliquots
Prepare buffer and fixative aliquots
Prepare Phosphate Buffer (PB) aliquots for transfer to the clinic
Prepare PB Solution X by dissolving
EITHER
14.91 g of Na2HPO4 (anhydrous) in 525 mL of ddH2O
OR
18.69 g of Na2HPO4•2H2O (dihydrate) in 525 mL of ddH2O
Filter sterilise and store at 4 °C
Prepare PB Solution Y by dissolving
EITHER
13.80 g of NaH2PO4•H2O (monohydrate) in 500 mL ddH2O
OR
15.60 g of NaH2PO4•2H2O (dihydrate) in 500 mL ddH2O
Filter sterilise and store at 4 °C
Prepare 0.2 Molarity (M) PB 7.4
- Mix 405 mL of PB Solution X with 95 mL of PB Solution Y
- Adjust the pH to 7.4 using hydrochloric acid (HCl)
- Filter sterilise and store at 4 °C
Prepare 25 x 22.2 mL aliquots of 0.113 Molarity (M) PB 7.4
- Mix 450 mL of filter sterilised0.2 Molarity (M) PB 7.4 with 349.2 mL of ddH2O in sterile glassware
- Using a sterile 25 mL serological pipette, add 22.2 mL of 0.113 Molarity (M) PB to each 30 mL polypropylene universal tube
- Label each tube with contents and date and store at 4 °C
Prepare 25 x 2.8 mL aliquots of 36% EM-grade formaldehyde (FA) Safety Critical
Using a sterile 5 mL serological pipette, add 2.8 mL of 36% FA to each 5 mL amber centrifuge tube Safety Critical
Label each tube, place in a labelled box which clearly displays the hazard warning symbols, and store at Room temperature Safety Critical
Transfer buffer and fixative aliquots to the clinic
Collection of biopsies in EM-grade fixative
Collection of biopsies in EM-grade fixative
Prepare a vial of EM-grade fixative for each biopsy on the day of sample collection Safety Critical
Note
Normally this should be done in a fume hood. In a clinical setting, a fume hood may not be available. If this is the case, follow local rules, and take care to open the FA vial for the minimal time required to dispense it into the PB vial.
For each biopsy, add one FA aliquot to one PB aliquot Safety Critical
This will make a vial of 4 % (v/v) FA in 0.1 Molarity (M) PB
Prior to tissue collection, ensure that the appropriate labelled vial containing primary fixative is at hand so that time between tissue collection and fixation is minimised.
Immediately after collection, add the biopsy to the fixative vial. The fixative vial should only be opened for the time needed to allow insertion of the sample. PPE should be worn Safety Critical
Note
If the sample cannot be immediately immersed in fixative at the bedside or in theatre, then it should be immersed as soon as safe and practical to do so, for example, on arrival in the pathology lab. However, it is critical to note that any drying of the tissue will result in deterioration of the ultrastructure.
Fix biopsy for at least 12:00:00 at Room temperature
Note
We suggest that the biopsy is fixed for 12 h at room temperature to help ensure that any unknown pathogens in the sample are inactivated. The biopsy should then be stored in the fridge at 4ºC until further processing.
12h
Long term storage of fixed biopsies
Long term storage of fixed biopsies
Transfer samples to a vial containing at least 1 mL of 1 % (v/v) FA in 0.1 Molarity (M) PB and store at 4 °C .
Note
We suggest that the storage fixative be replaced with freshly made 1% FA in 0.1 M PB every 2 to 3 months. Formaldehyde is known to undergo slow reactions including oxidisation to formic acid. Polymerisation may also occur with associated reduction in the availability of formaldehyde monomers. The temporal kinetics of these reactions in a buffered system of 1% FA at 4ºC appears to be unknown.