# Onion Peel Powder's Impact on the Leptin Receptors in the Hippocamp of Obese Rats
## 2 Materials and Methods
### Immunolistochemistry and Antibodies  
After euthanasia (decapitation), the brains were immediately stored in a 10% solution of buffered formalin (at pH = 7). After 12 h of storage at 4 \({}^{\circ}\)C, the brains were dehydrated in increasing concentrations of ethyl alcohol. As described previously [25], the brains were finally embedded in paraffin blocks and cut with a microtome into 5 \(\upmu\)m thick sections (Microm HM 360, Microm, Walldorf, Germany). The regions of Bregma ranging from \(-\)2.5 mm to \(-\)3.8 mm of the rat brain were analyzed. The brain sections were placed on adhesive glass slides (Superfrost Plus, Thermo Scientific, Braunschweig, Germany) and used for further immunohistochemical staining using the peroxidase anti-peroxidase method (PAP) according to the following protocol: First, to remove paraffin, the sections were washed in xylene three times for 15 min each. Then, the slides were hydrated via sequential incubation with a graded series of ethyl alcohol, and in the end, they were washed in distilled water. In the following step, the slides were put in a container containing citrate buffer (at pH = 6) and heated three times for 7 min to 97 \({}^{\circ}\)C using a microwave (800 W) to retrieve antigens. Then, the selected brain sections were outlined with a hydrophobic marker (ImmEdgeTM Hydrophobic Barrier Pen, Vector Laboratories, Burlingame, CA, USA). In order to block the endogenous peroxidase activity, the sections were chilled and washed in 3% hydrogen peroxidase for the following 20 min. The brain slides were then flushed twice with PBS solution at pH = 7.4 (each time for 15 min) and subjected to incubation in 2.5% normal goat serum (ImPRESSTM, MP-7451, Vector Labs, Burlingame, CA, USA) at room temperature (RT) for the next 20 min. Then, one part of the sections was incubated for 24 h at 4 \({}^{\circ}\)C with primary polyclonal rabbit antibodies raised against leptin receptor LepR (1:200, DF7139, Biorbyt, BIOKOM, Falenty, Poland), while the second part was incubated with primary rabbit anti-glial fibrillary acidic protein \begin{table}
\begin{tabular}{|l|c|} \hline \multicolumn{1}{|c|}{**Parameters**} & \multicolumn{1}{c|}{**Onion Skin**} \\ \hline Moisture [\% d.m.] & 9.89 \(\pm\) 0.10 \\ \hline Protein [\% d.m.] & 2.58 \(\pm\) 0.10 \\ \hline Fat [\% d.m.] & 0.77 \(\pm\) 0.03 \\ \hline Ash [\% d.m.] & 5.50 \(\pm\) 0.18 \\ \hline Carbohydrate * [\% d.m.] & 19.17 \(\pm\) 0.99 \\ \hline TDF [\% d.m.] & 62.09 \(\pm\) 0.77 \\ \hline IDF [\% d.m.] & 54.71 \(\pm\) 0.62 \\ \hline SDF [\% d.m.] & 7.38 \(\pm\) 0.59 \\ \hline Energy [kcal/100 g] & 218.10 \(\pm\) 2.22 \\ \hline Total polyphenol [mg GAE/g d.m.] & 34.73 \(\pm\) 0.60 \\ \hline Total flavonoids [mg QE/g d.m.] & 41.81 \(\pm\) 4.83 \\ \hline PAC [\%/g d.m.] & 28.65 \(\pm\) 3.14 \\ \hline Q [mg/g d.m.] & 18.74 \(\pm\) 0.14 \\ \hline Q 4\({}^{\prime}\) G [mg/g d.m.] & 4.99 \(\pm\) 0.18 \\ \hline DPPH [mm TE/g d.m.] & 131.53 \(\pm\) 2.09 \\ \hline FRAP [mm TE/g d.m.] & 274.74 \(\pm\) 2.20 \\ \hline \end{tabular} Abbreviations: d.m.–dry matter; TDF—total dietary fiber; IDF—soluble dietary fiber; GAE—solid equivalent; QE—quercetin equivalent; PAC—total phenolic acid content; Q—quercetin; Q * \({}^{d}\)G—quercetin glucoside; DPPH—radical scavenging ability against1,1-diphenyl-2-picrylhydrazryl; FRAP—ferric reducing antioxidant potential; TE—trolox equivalent. * Available carbohydrates, calculated by subtracting total dietary fiber from total content of carbohydrates.  
\end{table} 
Table 2: Chemical composition and antioxidant properties of onion skin powder [13; 23]. (GFAP) antibody (G9269, 1:80, Sigma Aldrich, St. Louis, MO, USA) overnight at 4 \({}^{\circ}\)C. After this procedure, the slides were washed two times for 15 min using a washing buffer and covered for 1 h with the anti-rabbit Ig (ImPRESSTM, MP-7451 Vector Labs, Burlingame, CA, USA). The 3.3\({}^{\prime}\)-diaminobenzzidine chromogen was used to visualize primary antisera (ImmPACTTM DAB, SK-4105, Vector Labs, Burlingame, CA, USA).