Feb 25, 2020

Public workspaceHuBMAP - Tissue Processing with Paraffin Infiltration

DOI
dx.doi.org/10.17504/protocols.io.8nzhvf6
  • 1University of Florida
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: Jeff.spraggins@vanderbilt.edu
Marda Jorgensen
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DOI: dx.doi.org/10.17504/protocols.io.8nzhvf6
Protocol Citation: Marda Jorgensen, Franchesca Farris, Jerelyn Nick 2020. HuBMAP - Tissue Processing with Paraffin Infiltration. protocols.io https://dx.doi.org/10.17504/protocols.io.8nzhvf6
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: October 23, 2019
Last Modified: October 02, 2020
Protocol Integer ID: 29113
Keywords: histology, codex, microtomy, tissue sectioning, tissue, pathology, research specimens, histopathology, hubmap, embedding, tissue processing, processing, vip
Abstract
The purpose of this Standard Operating Procedure (SOP) is to outline procedures for the automated processing of formalin fixed HuBMAP specimens for paraffin embedding.
Guidelines
  • Managers and supervisors - are responsible for making sure that technicians are properly trained and equipment and facility are maintained in good working order.
  • Laboratory personnel - are responsible for reading and understanding this SOP and related documents and to perform these tasks in accordance with the SOPs.
Materials
MATERIALS
ReagentParaplastCatalog #39501006
Reagent100% EtOHGold Shield ChemicalsCatalog #DSP-CA-151
ReagentXyleneFisher ScientificCatalog #X3P-1GAL
ReagentSakura® VIP 6 Tissue ProcessorSakura FinetekCatalog #VIP 6
ReagentTissue Cassette BasketNewcomer SupplyCatalog ## 5112D
10% Neutral Buffered Formalin (NBF)
Paraffin Wax (Paraplast X-tra)
Xylene
Ethanols (EtOH)- 70%, 95%, 100%
Tissue Processor (automatic)
Tissue Processing Cassette Basket

Safety warnings


  • As these specimens are both human in origin and emmersed in formaldehyde, gloves and other PPE are required.
  • Chemicals used during processing are flammable (ethanol and xylenes).Do not overfill reagent bottles.
  • Paraffin melting point cannot exceed 70C.
  • Oven and paraffin containers are hot, use extreme care.
  • Never open retort lid if vacuum or pressure is being displayed- open only when a,mbient is displayed.
Before start
  • Confirm processor is on, and reagent bottles are filled to specified volumes (indivxcated on labels).
  • Check that regent bottle screw caps are tightened
Ensure that the retort is drained and the Clean Cycle has been run. If not, perform Clean Cycle.
Processing Selection & Initial Steps
Processing Selection & Initial Steps
Place dividers in processor sample basket and load cassettes. No more than 10 cassettes can be added to each partition. Cover the basket with the basket lid.

Note
Perform transfer of cassettes in the hood to avoid inhalation of the 70% ethanol that the cassettes are held in prior to procesing

1d
Open the retort and place the basket inside. Rotate handles until they lay flat against the sides of the chamber. Close and latch retort
1d
Press "Tissue Processing" on Main Menu. Enter desired program number (program used listed below). Select Immediate Start or Delayed Start to control the begining of processing so that completion coincides wih availability to embed at termination.


Note
Appendix 1
Paraffin Fixation and Processing Program


Station # Solution* Conc. Time Set Temp. P/Vacuum Mixer
1 EtOH 70% Hold** _________ off off
2 EtOH 70% 0:35 _________ P/V slow
3 EtOH 80% 0:35 _________ P/V slow
4 EtOH 95% 0:35 _________ P/V slow
5 EtOH 95% 0:35 _________ P/V slow
6 EtOH 100% 0:35 _________ P/V slow
7 EtOH 100% 0:35 _________ P/V slow
8 Xylene 100% 0:45 _________ P/V slow
9 Xylene 100% 0:45 _________ P/V slow
10 Paraffin _________ 0:45 60⁰C P/V slow
11 Paraffin _________ 0:45 60⁰C P/V slow
12 Paraffin _________ 0:45 60⁰C P/V slow
13 Paraffin _________ 0:45 60⁰C P/V slow

*Maintenance if performed according to manufacturer’s recommendation.
**Duration of holding time dependent on desired end time to start embedding.

3d
When processing is completed remove basket and press "Drain Retort"
Transfer cassettes from the processing basket to the embedding station paraffin chamber.