Literally a quick reference of the lab's standard protocols/items.

<aside> 👉 When doing multiple labels, be careful to check that the fluorophores are compatible using the Fluorescence SpectraViewer.

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Table of Contents

TRG Go-to Labels

Wisteria Floribunda agglutinin (WFA)

(from Morikawa et al., 2017, Nat Sci Reports)

<aside> 🖇 "Primary": Biotin-conjugated WFA (1:3,000; BA-3101, EY laboratories) "Secondary": Alexa ###-conjugated streptavidin (1:1000 for Alexa 647 (catalog number S32357), 1:500 for Alexa 594 (catalog number S11227); 1:500 for Alexa 488)

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1. Fix: Perfused with 4% PFA/PBS. (Post-fix: 4% PFA/PBS 4 degreeC O/N → 30% sucrose/PBS 4 degreeC 2 O/N) Free-floating coronal section (30 μm) using a cryostat.
2. Blocking: 5% BSA/PBS/0.3%Triton X-100 at RT for 1 h.

<aside> ⚠️ CAUTION FOR WFA: Bovine Serum Albumin or Normal Goat Serum can contain carbohydrates that interfere with the lectin label. For these reactions, use a carbo-free blocking solution instead (Cat#: SP-5040-125 Vector Labs). You can even dilute your lectin tag in here instead.

(The 5% BSA/PBS/0.3%Triton X-100 and 5% Normal Donkey Serum/PBS/0.3%TritonX-100 have worked for us in the past, but the results are not as consistent for WFA. NDS solutions have worked best when we use WFA with antibodies).

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1. Lectin-Reaction: Biotin-conjugated WFA (1:3,000; BA-3101, EY laboratories) in 1% BSA/PBS/0.3% Triton X-100 at 4 degree C overnight.
2. Wash: PBS at 3 times for 5min.
3. Biotin-Avidin reaction: Alexa ###-conjugated streptavidin (1:500 for Alexa 594; 1:1000 for Alexa 647) in 1% BSA/PBS/0.3% Triton X-100 at RT for 90 min.
4. Wash: PBS at 4 times for 10 min.
5. Mounting Solution: Prolong Diamond Antifade Reagent containing DAPI.

cfos

(from Morikawa et al., 2017, Nat Sci Reports and Ramirez Lab)

<aside> 🖇 Primary: Rabbit polyclonal anti-c-Fos antibody (1:5,000; #226003, Synaptic Systems) Secondary: Alexa ###-conjugated donkey anti-rabbit IgG (1:1,000 for Alexa 488 or 1:200 for Alexa 555; Thermo)

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