# Protocols **General** - Koehler illumination and DIC configuration for [inverted](/file_url/208) and [upright](/file_url/209) microscopes - All HCBI coverslip corrected objectives are corrected for **\#1.5 Coverslips**. Please ensure you are ONLY using #1.5 slips. - The HCBI recomends Prolong Glass (hard mount) or Slow Fade Glass (soft mount) mounting media. We do NOT recomend using moutning media with DAPI pre-mixed into it. This causes substantial background. Nuclear dyes should be applied with/after your secondary antibodies and washed extensively before mounting. ### **Lightsheet** - [Lightsheet consumables](/file_url/501)(HCBI recommended suppliers) - [Guide to Lightsheet sample prep](/file_url/172) (Zeiss) - Click [here](http://www.youtube.com/watch?v=3KIMDtdq_1c) to view a video of Lightsheet sample prep (Zeiss) - [Guide to stitching tiles with Arivis software](/file_url/243) ### **MERSCOPE** 1. Gene Panel Design - This is completed on the Vizgen website and is at minimum a 4-6 week process. A guide to the Gene panel Design can be found [here](/file_url/373). 2. The HCBI does NOT provide any reagents or equipment required for sample prep. A list of all required general lab reagents and equipment can be found [here](/file_url/376). 3. A list of reagents/equipment to order from Vizgen can be found [here](/file_url/375) (from Dec 2021, prices subject to change). 4. The sample prep protocol can be found [here](/file_url/374). 5. A video introduction to the Vizgen Vizualizer software can be found [here](https://youtu.be/N2WYfBHtlz8). ### **Tissue Clearing** - [Clearing with CLARITY and the X-CLARITY ETC system](/file_url/264) - [Immunostaining for cleared tissue](/file_url/297) (from Renier et al., Cell, 2016) **Axio Scan** - [Axio Scan Application Guide](/file-url/181) ### **Bruker Lightsheet** - [User Manual](/file_url/452) ### **Expansion Microscopy** - [Boyden Lab Protocols](https://expansionmicroscopy.org/) - [Chambers for Gelation](https://www.emsdiasum.com/secure-seal-hybridization-chambers) ### **ELYRA** - [Multi-color channel alignment](/file_url/205) - [PALM/STORM sample prep](/file_url/122) ### **STEDYCON** [STED Sample Preparation and Labeling Guide](/file_url/479) ### **PALM Laser Microdissection** - [Immunohistochemistry on frozen sections prior to microdissection and RNA extraction](/file_url/117) - [Sample staining, mounting, and cutting for RNA extraction](/file_url/124) - [Quickguide to PALM microdissection consumables](/file_url/118) - [RNA extraction from formalin fixed paraffin embedded (FFPE) sections](/file_url/120) - [RNA extraction from frozen sections](/file_url/121)[Immunofluorescence on frozen sections](/file_url/119) ### **LSM 780/880** - [Guide to FCS experiments](/file_url/123) ### ZEN - [Tiling and focusing with ZEN Blue](/file_url/207) --- Attachments- [ picture\_as\_pdf lightsheet\_consumables.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/lightsheet_consumables_0.pdf) - [ picture\_as\_pdf lightsheetz1\_sample-preparation\_zeiss.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/lightsheetz1_sample-preparation_zeiss.pdf) - [ picture\_as\_pdf ihc\_and\_rna.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/ihc_and_rna.pdf) - [ description palm\_consumables\_quickguide\_2012\_rev\_d.docx ](/sites/g/files/omnuum7436/files/hcbidoug/files/palm_consumables_quickguide_2012_rev_d.docx) - [ picture\_as\_pdf bruker\_user\_manual.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/bruker_user_manual.pdf) - [ picture\_as\_pdf zeiss\_labprotocol\_immunofluorescence\_0310.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/zeiss_labprotocol_immunofluorescence_0310.pdf) - [ picture\_as\_pdf zeiss\_lcm\_protocol\_rna\_ffpe\_0212.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/zeiss_lcm_protocol_rna_ffpe_0212.pdf) - [ picture\_as\_pdf zeiss\_lcm\_protocol\_rna\_frozen\_0212.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/zeiss_lcm_protocol_rna_frozen_0212.pdf) - [ picture\_as\_pdf en\_41\_011\_065\_elyra\_sample-prep-quickguide.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/en_41_011_065_elyra_sample-prep-quickguide.pdf) - [ picture\_as\_pdf elyra\_sim\_channel\_alignment\_protocol.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/elyra_sim_channel_alignment_protocol.pdf) - [ picture\_as\_pdf guide\_to\_basic\_fcs\_experiment.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/guide_to_basic_fcs_experiment.pdf) - [ picture\_as\_pdf zeiss\_labprotocol\_rna\_0811.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/zeiss_labprotocol_rna_0811.pdf) - [ picture\_as\_pdf axio\_scan.z1\_application\_guide.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/axio_scan.z1_application_guide.pdf) - [ picture\_as\_pdf tile\_imagingfocus\_faq\_rev\_03-14.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/tile_imagingfocus_faq_rev_03-14.pdf) - [ picture\_as\_pdf aim\_zen2012\_aim\_dic\_inverted\_lsm.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/aim_zen2012_aim_dic_inverted_lsm.pdf) - [ picture\_as\_pdf aim\_zen2012\_aim\_dic\_upright\_lsm.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/aim_zen2012_aim_dic_upright_lsm.pdf) - [ picture\_as\_pdf en\_quick-guide\_lightsheet-z.1\_arivis-stitching.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/en_quick-guide_lightsheet-z.1_arivis-stitching.pdf) - [ picture\_as\_pdf hcbi\_clarity\_protocol.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/hcbi_clarity_protocol.pdf) - [ picture\_as\_pdf immunostaining\_protocol.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/immunostaining_protocol.pdf) - [ picture\_as\_pdf Gene\_Panel\_Design ](/sites/g/files/omnuum7436/files/hcbidoug/files/a_guide_to_designing_your_merfish_gene_panel_final.pdf) - [ picture\_as\_pdf 91600002\_merscope\_fresh\_and\_fixed\_frozen\_tissue\_sample\_preparation\_user\_guide\_rev\_b.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/91600002_merscope_fresh_and_fixed_frozen_tissue_sample_preparation_user_guide_rev_b.pdf) - [ list\_alt merscope\_reagent\_product\_catalog\_december\_2021.xlsx ](/sites/g/files/omnuum7436/files/hcbidoug/files/merscope_reagent_product_catalog_december_2021.xlsx) - [ picture\_as\_pdf lab\_setup\_check\_list\_.pdf ](/sites/g/files/omnuum7436/files/hcbidoug/files/lab_setup_check_list_.pdf) - [ picture\_as\_pdf sted\_sample\_preparation\_and\_labelling.pdf ](/sites/g/files/omnuum7436/files/sted_sample_preparation_and_labelling.pdf) ---